Methods and systems for treating or preventing cancer

ABSTRACT

The present invention generally relates to compositions and methods for treatment of subjects having or at risk of cancer or other conditions. In some cases, the composition may include nitric oxide and/or peptides such as thyrotropin-releasing hormone (TRH) and/or GnRH (gonadotropin-releasing hormone). The nitric oxide and/or peptide may be present within a first phase comprising a lecithin, such as phosphatidylcholine. In certain embodiments, the lecithin is present in liposomes, micelles, or other structures containing nitric oxide. The composition can take the form of a gel, a cream, a lotion, an ointment, a solution, a solid “stick,” etc., that can be rubbed or sprayed onto the skin. Other aspects of the present invention are generally directed to methods of making or using such compositions, methods of promoting such compositions, kits including such compositions, or the like.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/801,188, filed Mar. 13, 2013, entitled “Methods and Systemsfor Treating or Preventing Cancer,” by Perricone. This application isalso a continuation-in-part of U.S. patent application Ser. No.13/801,402, filed Mar. 13, 2013, entitled “Systems and Methods forDelivery of Peptides,” by Perricone. Each of these is incorporatedherein by reference in its entirety.

FIELD

The present invention generally relates to compositions and methods fortransdermal delivery, including treatment of cancer and otherconditions.

BACKGROUND

Cancer is a group of diseases that involves unregulated cell growth.Cancer is usually treated with chemotherapy, radiation therapy, and/orsurgery. Survival rates vary by the type and location of the cancer andthe extent of the disease. While cancer can occur at any age (and somecancers are more common in children), the risk of developing cancergenerally increases with age. Cancer rates are currently rising, partlydue to increased life expectancies throughout the world.

SUMMARY

The present invention generally relates to compositions and methods fortransdermal delivery, including treatment of cancer and otherconditions. The subject matter of the present invention involves, insome cases, interrelated products, alternative solutions to a particularproblem, and/or a plurality of different uses of one or more systemsand/or articles.

In one aspect, the present invention is generally directed to a method.According to one set of embodiments, the method comprises an act ofadministering, to the skin of a subject having or at risk of cancer, acomposition comprising an effective amount of a peptide and nitric oxideto treat or prevent the cancer, and a carrier having aphosphatidylcholine component entrapping the nitric oxide.

In accordance with another set of embodiments, the present invention isgenerally directed to a method comprising contacting the skin of asubject having or at risk of cancer with a composition comprising anemulsion comprising a first phase comprising a peptide, nitric oxide,and lecithin, and a second phase comprising an emulsifier. In somecases, the lecithin is present at least about 0.25% by weight of thecomposition, and in some embodiments, the first phase comprises no morethan about 250 ppm of water by weight of the composition.

According to still another set of embodiments, the method comprises anact of administering, to the skin of a subject having or at risk ofcancer, a composition comprising an effective amount ofgonadotropin-releasing hormone (GnRH) and nitric oxide to treat orprevent the cancer, and a carrier having a phosphatidylcholine componententrapping the nitric oxide.

The method, in another set of embodiments, comprises contacting the skinof a subject having or at risk of cancer with a composition comprisingan emulsion comprising a first phase comprising gonadotropin-releasinghormone (GnRH), nitric oxide, and lecithin, and a second phasecomprising an emulsifier, wherein the lecithin is present at least about0.25% by weight of the composition, and wherein the first phasecomprises no more than about 250 ppm of water by weight of thecomposition.

The method, in yet another set of embodiments, includes administering,topically to the skin of a subject, a composition comprising aneffective amount of gonadotropin-releasing hormone (GnRH) to a subjecthaving or at risk of cancer.

In another set of embodiments, the method includes administering,topically to the skin of a subject, a composition comprising aneffective amount of thyrotropin-releasing hormone (TRH) to treat asubject having or at risk of cancer.

In another aspect, the present invention encompasses methods of makingone or more of the embodiments described herein, for example, acomposition comprising nitric oxide. In still another aspect, thepresent invention encompasses methods of using one or more of theembodiments described herein, for example, a composition comprisingnitric oxide.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention. In cases where the present specificationand a document incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows results from the laser Doppler assay in four humanstudy participants.

DETAILED DESCRIPTION

The present invention generally relates to compositions and methods fortreatment of subjects having or at risk of cancer or other conditions.In some cases, the composition may include nitric oxide and/or peptidessuch as thyrotropin-releasing hormone (TRH) and/or GnRH(gonadotropin-releasing hormone). The nitric oxide and/or peptide may bepresent within a first phase comprising a lecithin, such asphosphatidylcholine. In certain embodiments, the lecithin is present inliposomes, micelles, or other structures containing nitric oxide. Thecomposition can take the form of a gel, a cream, a lotion, an ointment,a solution, a solid “stick,” etc., that can be rubbed or sprayed ontothe skin. Other aspects of the present invention are generally directedto methods of making or using such compositions, methods of promotingsuch compositions, kits including such compositions, or the like.

According to one aspect of the present invention, a composition asdescribed herein is used to treat cancer or other conditions. To “treat”a disorder means to reduce or eliminate a sign or symptom of thedisorder, to stabilize the disorder, and/or to reduce or slow furtherprogression of the disorder. Thus, in one set of embodiments, theapplication of nitric oxide and/or peptide, e.g., in a nitric oxideand/or peptide matrix, to the skin may result in clinical improvement.In some cases, the delivery of nitric oxide and/or peptide to the skin,e.g., to the epidermis and/or dermis, may be achieved at a controlledrate and/or concentration. In other embodiments, a composition describedherein is administered as an adjuvant to traditional chemotherapy.Without wishing to be bound by any theory, it is believed that nitricoxide is effective in treating cancer, such as prostate cancer, asevidenced by studies with nitric oxide donors such as nitroglycerin. Inaddition, nitric oxide can enhance cytotoxic efficacy of somechemotherapeutic agents and radiation.

According to another aspect of the present invention, compositionscomprising peptides provided herein are effective in treating cancer,alone or in combination, e.g. in a common composition with, nitricoxide. In some cases, the peptide may include two or more amino acidslinked by the carboxyl group of one amino acid to the amino group ofanother, e.g. a peptide linkage, to form an amino acid sequence. It iscontemplated that peptides may be purified and/or isolated from naturalsources or prepared by recombinant or synthetic methods. Amino acidsequences may be encoded by naturally or non-naturally occurring nucleicacid sequences or synthesized by recombinant nucleic acid sequences orartificially synthesized. A peptide may be a linear peptide or acyclopeptide, e.g. cyclic including bicyclic. In some cases, a “peptide”may be interchangeably referred to as a “therapeutic peptide.” In somecases, the peptide is a “pseudo-peptide” or a “peptidomimetic,” whichare compounds designed to functionally mimic a peptide. Non-limitingexamples of peptides include thyrotropin-releasing hormone (TRH),melanocyte-stimulating hormone (MSH), or gonadotropin-releasing hormone(GnRH).

Non-limiting examples of peptides, pseudo-peptides, or peptidomimeticscontemplated by the present invention include melanocyte-stimulatinghormones (e.g. α-MSH, β-MSH, and γ-MSH, collectively “MSH”)pro-opiomelanocortin (POMC), thyrotropin-releasing hormone (TRH),buserelin, goserelin, histrelin, leuprolide, leuprorelin, triptorelin,abarelix, degarelix, ADH-1 (Exherin®), bortezomib, macrophage-activatinglipopeptide-2 (MALP-2), thymopentin, cilengitide, tripeptide RGD,ATN-161 (Ac-PHSCN-NH₂), tumstatin peptide (TLPFAYCNIHQVCHYAQRNDRSYWL)(SEQ ID NO: 1), tumstatin fragment (YSNSG) (SEQ ID NO: 2), pentastatin-1(LRRFSTMPFMFCNINNVCNF) (SEQ ID NO: 3), endostatin peptide(HTHQDFQPVLHLVALNTPLSGGMR) (SEQ ID NO: 4), endostatin frament IV(CETWRTETTGATGQASSLLSGRLLEQKAASCHNSYIVLCIENSFMTSFSK) (SEQ ID NO: 5),endostatin frament I (FLSSRLQDLYSIVRRADRAA) (SEQ ID NO: 6), C16Y(DFKLFAVYIKYR) (SEQ ID NO: 7), C16S (DFKLFAVTIKYR) (SEQ ID NO: 8), VEGFderived peptides (e.g. _(D)(LPR) and KSVRGKGKGQKRKRKKSRYK (SEQ ID NO:9)), FGF-derived peptides (e.g. Ac-ARPCA), P144 (TSLDASIIWAMMQN) (SEQ IDNO: 10), HPRG derived peptides (e.g. (HHPHG)₄ (SEQ ID NO: 11)), KV11(YTMNPRKLFDY) (SEQ ID NO: 12), fibrinogen derived peptides (e.g.ARPAKAAATQKKVERKAPDA) (SEQ ID NO: 13), PF4 derived peptides (e.g.NGRKISLDLRAPLYKKIIKKLLES) (SEQ ID NO: 14), chemokinostatin-1(NGREACLDPEAPMVQKIVQKMLKG) (SEQ ID NO: 15), anginex(ANIKLSVQMKLFKRHLKWKIIVKLNDGRELSLDA) (SEQ ID NO: 16), DI-TSPa (GVITRIR)(SEQ ID NO: 17), properdistatin (GPWEPCSVTCSKGTRTRRR) (SEQ ID NO: 18),PEDF-TGA fragments (e.g. TGALVEEEDPF (SEQ ID NO: 19) andDPFFKVPVNKLAAVSNFGYDLYRVRSSMSPTTN (SEQ ID NO: 20)), PEDF P18(NFGYDLYRVRSSTSPTTN) (SEQ ID NO: 21), SvOrth-2(NVLLSPLSVATALSALSLGAEQRTES) (SEQ ID NO: 22), pTnI(EDMNQKLFDLRGKFKRPPLRRVRMSADAML) (SEQ ID NO: 23), PAMP₁₂₋₂₀ (KWNKWALSR)(SEQ ID NO: 24), IM862 (EW), Aβ (EVHHQKLVFF) (SEQ ID NO: 25), as well assalts or analogs of any of the foregoing.

In some embodiments, compositions comprising MSH (e.g. α-MSH) providedherein are effective in treating cancer, alone or in combination, e.g.in a common composition with, nitric oxide. Without wishing to be boundby any particular theory, it is believed that MSH, signaling through amelanocortin-1 receptor, augments antitumoral immunity by up-regulatingthe expression of cytotoxic genes and enhancing the cytolytic activityin tumor-specific CD8⁺ T cells, thereby resulting in a beneficialresponse for cancer treatment, e.g. melanoma. Thus, the beneficialeffects of nitric oxide discussed herein may work synergistically, oradditively, with MSH, e.g. by promoting increased availability,delivery, and/or circulation of MSH in a subject. In some aspects, theMSH used in a composition comprising MSH is derived from apro-opiomelanocortin (POMC) precursor poplypeptide. In some aspects, theMSH used in a composition comprising MSH is synthesized. Non-limitingexamples of MSH contemplated by the present invention include α-MSH,β-MSH, γ-MSH, afamelanotide (melanotan-1), bremelanotide, melanotan II,as well as salts and analogs of any of the foregoing.

In some embodiments, as discussed herein, compositions are provided thattreat the symptoms of cancer. In one example, compositions comprisingTRH provided herein are effective in treating cancer-related fatigue,alone or in combination, e.g. in a common composition with, nitricoxide. Fatigue is a common and often disabling symptom for cancerpatients. Without wishing to be bound by theory, it is believed thatTRH, a key regulator of homeostasis, exerts arousing and analepticactions, which reduce cancer-related fatigue and increase the quality oflife for subjects having cancer-related fatigue. Thus, compositionscomprising TRH and/or nitric oxide may positively impact cancer-relatedfatigue in subjects having cancer-related fatigue by promoting arousal.In addition, the beneficial effects of nitric oxide discussed herein maywork synergistically, or additively, with TRH, e.g. by promotingincreased availability, delivery, and/or circulation of TRH in asubject. In some embodiments, the composition comprises an analog ofTRH. Non-limiting examples of TRH or TRH analogs contemplated by thepresent invention include protirelin (Thyrel TRH, Relafect TRH),taltirelin (Ceredist), montirelin, as well as salts and analogs of anyof the foregoing.

Without wishing to be bound by any theory, it is believed that TRH isinvolved in energy metabolism. TRH may also be involved in mitochondrialactivity and metabolic capacity. Thus, in some cases, TRH can be used totreat cancer or cancer fatigue. As mentioned, TRH can be readilyobtained from commercial sources. As is known by those of ordinary skillin the art, TRH has the structure pyroGlu-His-Pro-NH₂ or:

In another set of embodiments, the composition comprises nitric oxideand/or gonadotropin-releasing hormone (GnRH), or an analog thereof. Thecomposition may also comprise other peptides, such as MSH or TRH, or anyother peptide described herein, in some embodiments. GnRH is also knownas also known as luteinizing-hormone-releasing hormone (LHRH) orluliberin. As is known to those of ordinary skill in the art, GnRH hasthe structure pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (SEQ IDNO: 26), and GnRH and its analogs can be readily obtained fromcommercial sources. Non-limiting examples of GnRH or GnRH analogsinclude gonadorelin hydrochloride (Factrel®), gonadorelin diacetatetetrahydrate (Cystorelin®), leuprolide, leuprolide acetate, deslorelinas well as salts and analogs of any of the foregoing.

GnRH is a trophic peptide hormone that has multiple effects, includingrelease of follicle-stimulating hormone (FSH) and luteinizing hormone(LH) from the anterior pituitary. It is typically produced and releasedendogenously from neurons within the hypothalamus. In addition, in somecases, cancers that are hormonally sensitive may be treated using GnRH.Examples include, but are not limited to, prostate cancer or breastcancer. GnRH may also have an effect on mitochondrial activity in somecells, for example, increasing GnRH exposure may include the number ofmitochondria in cells and/or the activity level of the mitochondria.

Accordingly, without wishing to be bound by any theory, it is believedthat the application of GnRH, e.g., within a composition as discussedherein, may be used to treat a person for having or at risk of cancer,etc. For example, a composition as discussed herein may be applied tothe skin of a subject, which may lead to the treatment or prevention ofcancers, such as those discussed herein. In some cases, a composition asdiscussed herein may also include NO, e.g., along with a carrier havinga phosphatidylcholine component entrapping the nitric oxide, asdiscussed herein. However, as discussed herein, in some cases, thecomposition does not necessarily include NO.

In one set of embodiments, compositions of the present invention may beused to facilitate the transport of GnRH (and/or other peptides such asis discussed herein) across the blood-brain barrier into the brain,e.g., into the hypothalamus, and/or to other areas of the brain. Withoutwishing to be bound by any theory, it is believed that in someembodiments, the application of compositions comprising GnRH (and/orother peptides) to the skin, e.g., in a cream or other formulationcomprising NO, nitric oxide, causes the GnRH and the NO to enter thebloodstream, and the presence of NO may facilitate the transport of GnRH(and/or other peptides) across the blood-brain barrier.

In addition, in some embodiments, the composition may include both GnRHand TRH. Without wishing to be bound by any theory, it is believed thatthe combination of GnRH and TRH may have surprising and synergisticeffects in some cases. For example, both TRH and GnRH have effects onmitochondrial and metabolic activity within the body, and stimulation ofmitochondria by both TRH and GnRH may result in dramatic increases inthe number of mitochondria in cells and/or the activity level of themitochondria, compared to only TRH or only GnRH. In addition, TRH andGnRH may act on different systems within the body; for example, in oneset of embodiments, GnRH may be used to directly treat cancer cells,boost immune response, and/or increase metabolic levels, while TRH maybe used to treat fatigue or other effects of cancer. Thus, applicationof TRH and GnRH may provide synergistic effects for cancer treatment.Additionally, in some embodiments, the composition may further includenitric oxide and/or other peptides, e.g., as discussed herein.

Exemplary cancers include non-solid tumors such as leukemia, for exampleacute myeloid leukemia, multiple myeloma, hematologic malignancies orlymphoma, and also solid tumors and their metastases such as melanoma,non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma,glioblastoma, carcinoma of the thyroid, cholangiocarcinoma, bile duct,bone, gastric, brain/CNS, head and neck, hepatic, stomach, prostate,breast, renal, testicular, ovarian, cervix, skin, cervical, lung,muscle, neuronal, esophageal, bladder, lung, uterine, vulval,endometrial, kidney, colon, colorectal, pancreatic, pleural/peritonealmembranes, salivary gland, epidermoid tumors and hematologicalmalignancies. In certain embodiments, the cancer is prostate cancer. Incertain embodiments, the cancer is skin cancer (e.g., basal cellcarcinoma, squamous cell carcinoma, melanoma).

Thus, in one set of embodiments, application of nitric oxide and/orpeptides, e.g., in a penetrating matrix gel to deliver the active nitricoxide, may act as a preventative of and/or be used to treat cancer. Thecomposition may include any one or more of the peptides describedherein, with or without nitric oxide. In another set of embodiments, acomposition as described herein is applied to the skin of a subject,e.g., one having or at risk of cancer. For instance, in someembodiments, a composition as is described herein is contacted with theskin of a subject at a location where treatment of cancer (e.g., skincancer) is desired. Additionally, in some embodiments, the compositionmay be applied in conjunction with other types of treatments to asubject, e.g., to the skin of a subject, for treatment of any of thediseases, conditions, or needs described herein. These may be occur,e.g., simultaneously or sequentially, in various embodiments.

In some cases, the subject may be one that already has cancer. However,in other cases, the subject may not necessarily have cancer, but may beone that is at risk of developing cancer. The subject may also be onewith a family history of cancer in some instances.

In some embodiments, the nitric oxide and/or a peptide within thecomposition is stable at room temperature, and may remain active forextended periods of time, for example, for at least 1 year, at least 1.5years, at least 2 years, at least 2.5 years, at least 3 years, at least4 years, etc. The nitric oxide and the peptide may be released, forexample, when the composition is exposed to an aqueous environment,e.g., within the body. Without wishing to be bound by any theory, it isbelieved that when the composition is applied to the skin, the liquidcrystal structure collapses, delivering nitric oxide and/or the peptidesto, e.g., a muscle or other desired area of treatment. The concentrationof the nitric oxide and the peptide inside the liquid crystal matrix canbe varied in terms of concentration. The matrix also may act as asustained release and delivery system in some embodiments. It is alsobelieved that the liquid crystal is highly penetrating, such that nitricoxide and the peptide can be delivered to the epidermis, dermis anddermal vascular for systemic release as well as to subcutaneous fat, atleast under some conditions.

In accordance with some embodiments of the invention, nitric oxide gasitself may be entrapped or contained within various compositions asdiscussed herein, for example, in liquid crystal multilamellarphosphatidylcholine. In addition, in certain embodiments as discussedbelow, the composition may be stable and can be stored for periods oftime with little or no loss or reaction of the nitric oxide containedtherein.

Since nitric oxide is an unstable and reactive gas, entrapment, storage,and release of nitric oxide requires careful formulation in someembodiments of the invention. For example, nitric oxide readily reactswith water to form nitrous acid (HNO₂), and thus, certain embodiments ofthe invention include compositions or phases that are substantially freeof water. As another example, in one set of embodiments, nitric oxidemay be contained within a first phase comprising a lecithin such asphosphatidylcholine, which may be present within a second phasecomprising an emulsifier, such as is discussed herein. Other components,for example, transdermal penetration enhancers, adjuvants, surfactants,lubricants, etc. can also be present in certain cases.

Thus, the compositions of the invention comprise, in certain aspects, aphase comprising phosphatidylcholine and/or other lecithins in whichnitric oxide and/or peptides is/are contained within or “trapped.” Thephosphatidylcholine or lecithin may be contained within a second phase,for example, comprising an emulsifier, which may cause thephosphatidylcholine or lecithin to form vesicles, e.g., micelles orliposomes. The phosphatidylcholine or lecithin composition can beunilamellar or multilamellar in some embodiments. In some instances, thepresence of the second phase causes the phosphatidylcholine or lecithinto form a liquid crystal arrangement.

The nitric oxide is typically gaseous, and may be present within thecomposition as small bubbles and/or bound to lecithins orphosphatidylcholines within the composition. For example, the nitricoxide may be bound to double bonds present in the lecithins orphosphatidylcholines. Phosphatidylcholine is believed to stabilizeand/or contain the nitric oxide and/or peptides. In some cases,stability of the composition can be achieved at room temperature (about25° C.), and/or at other temperatures such as those described herein.Without wishing to be bound by any theory, it is believed that thephosphatidylcholine adopts a liquid crystal structure under suchconditions, which can thereby contain the nitric oxide, e.g., as smallgaseous bubbles, and/or through binding with lecithins orphosphatidylcholines. Similarly (without wishing to be bound by anytheory), it is contemplated that peptides are also contained within thevesicular and/or liquid crystal structure of phosphatidylcholine orother carriers described herein. In some aspects, (without wishing to bebound by any theory) the peptide, depending on its hydrophobicity, maybe contained within phosphatidylcholine vesicular membrane, or may betightly associated with the inner or outer surface of the membrane.

Nitric oxide is typically reactive with water (e.g., forming nitrousacid), which contributes to its relatively short lifetime within thebody or within other aqueous environments. Accordingly, in certainembodiments of the invention, the composition, or at least a phase ofthe composition comprising the nitric oxide (and/or the second phase,and/or one or more materials used to prepare a nitric oxide composition,and/or a nitric oxide composition prepared as described herein), issubstantially free of water, e.g., comprising no more than about 10 wt%, no more than about 3 wt %, no more than about 1 wt %, no more thanabout 0.3 wt %, or no more than about 0.1 wt % water (i.e., relative tothe weight of the overall composition). The composition may also have nomore than about 1,000 ppm, no more than about 750 ppm, no more thanabout 500 ppm, no more than about 400 ppm, no more than about 300 ppm,no more than about 250 ppm, no more than about 200 ppm, no more thanabout 150 ppm, no more than about 100 ppm, no more than about 50 ppm, nomore than about 25 ppm, or no more than about 10 ppm of water. Incertain embodiments, no detectable water may be present in thecomposition, or at least within a phase of the composition comprisingthe nitric oxide. Any suitable technique can be used for determining theamount of water present in the composition, for example, Karl-Fishertitration. In some cases, the composition may also be free of anyliquids that typically contain water, e.g., physiological buffers, bodyfluids, saline, or the like.

Any suitable amount of nitric oxide may be present within a compositionprepared as described herein. For example, at least about 0.3 wt %, atleast about 0.5 wt %, at least about 0.7 wt %, at least about 1 wt %, atleast about 1.5 wt %, at least about 2 wt %, at least about 2.5 wt %, atleast about 3 wt %, at least about 5 wt % at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, at least about 90 wt %, at least about 100 wt %, atleast about 110 wt %, or at least about 120 wt % of the composition canbe nitric oxide, where the basis of the weight percentage is the weightof the composition before nitric oxide is added. Combinations of any ofthese are also possible. For example, the nitric oxide may be present atbetween 70 wt % and about 120 wt % of the composition.

In some embodiments, the nitric oxide may be present at a concentrationof at least about 400 mg/kg, at least about 450 mg/kg, at least about500 mg/kg, at least about 550 mg/kg, at least about 570 mg/kg, at leastabout 600 mg/kg, at least about 650 mg/kg, at least about 700 mg/kg, atleast about 750 mg/kg, at least about 800 mg/kg, at least about 850mg/kg, at least about 950 mg/kg, or at least about 1000 mg/kg of thecomposition. In certain cases, the nitric oxide may be present at aconcentration of no more than about 2000 mg/kg, no more than about 1500mg/kg, no more than about 1000 mg/kg, no more than about 960 mg/kg, nomore than about 900 mg/kg, no more than about 800 mg/kg, no more thanabout 700 mg/kg, or no more than about 600 mg/kg. Combinations of any ofthese are also possible. For example, the nitric oxide may be present ata concentration of between about 570 mg/kg and about 960 mg/kg.

In some embodiments, the nitric oxide is present at a concentration(e.g., on a per-mass basis) of at least about 100 ppm, at least about200 ppm, at least about 300 ppm, at least about 400 ppm, at least about500 ppm, at least about 600 ppm, at least about 700 ppm, at least about800 ppm, at least about 900 ppm, at least about 1000 ppm, at least about1100 ppm, at least about 1200 ppm, at least about 1300 ppm, at leastabout 1400 ppm, at least about 1500 ppm, at least about 1600 ppm, atleast about 1700 ppm, at least about 1800 ppm, at least about 1900 ppm,at least about 2000 ppm, at least about 2500 ppm, at least about 3000ppm, at least about 3500 ppm, at least about 4000 ppm, at least about4500 ppm, at least about 5000 ppm, at least about 6000 ppm, at leastabout 7000 ppm, at least about 8000 ppm, at least about 9000 ppm, or atleast about 10000 ppm of the composition. In other embodiments, thenitric oxide is present at a concentration of no more than about 11000ppm, no more than about 10000 ppm, no more than about 9000 ppm, no morethan about 8000 ppm, no more than about 7000 ppm, no more than about6000 ppm, no more than about 5000 ppm, no more than about 4500 ppm, nomore than about 4000 ppm, no more than about 3500 ppm, no more thanabout 3000 ppm, no more than about 2500 ppm, no more than about 2000ppm, no more than about 1900 ppm, no more than about 1800 ppm, no morethan about 1700 ppm, no more than about 1600 ppm, no more than about1500 ppm, no more than about 1400 ppm, no more than about 1300 ppm, nomore than about 1200 ppm, no more than about 1100 ppm, no more thanabout 1000 ppm, no more than about 900 ppm, no more than about 800 ppm,no more than about 700 ppm, no more than about 600 ppm, no more thanabout 500 ppm, no more than about 400 ppm, or no more than about 300 ppmof the composition. Combinations of any of these are also possible. Forexample, in some embodiments, nitric oxide is present at a concentrationof between about 400 and about 900 ppm. NO content can be measured byany suitable technique. For example, in some embodiments, NO content ismeasured using a nitric oxide biosensor (e.g., nitric oxide macrosensorwith nitric oxide specific electrode from WPI Instruments). In someembodiments, NO content is measured by a change in weight in thecomposition after adding NO.

In some embodiments, nitric oxide is present within a first phasecomprising a lecithin, such as phosphatidylcholine. Phosphatidylcholine(herein abbreviated “PC”) is a basic component of cell membrane bilayersand the main phospholipid circulating in the plasma of blood.Phosphatidylcholine typically has a phospholipid structure with acholine head group and a glycerophosphoric acid tail group. The tailgroup can be saturated or unsaturated. More than one tail group may bepresent in the phosphatidylcholine in some cases, and the tail groupsmay be the same or different. Specific non-limiting examples ofphosphatidylcholines that could be used include one or a mixture ofstearic, palmitic, margaric, and/or oleic acid diglycerides linked to acholine ester head group.

Phosphatidylcholines are a member of a class of compounds calledlecithins. Typically, a lecithin is a composed of phosphoric acid,choline, fatty acids, glycerol, glycolipids, triglycerides, and/orphospholipids. In some cases, other lecithins may be used, in additionto or instead of a phosphatidylcholine. Non-limiting examples of otherlecithins include phosphatidylethanolamine, phosphatidylinositol, orphosphatidic acid. Many commercial lecithin products are available, suchas, for example, Lecithol®, Vitellin®, Kelecin®, and Granulestin®.Lecithin is widely used in the food industry. In some embodiments,certain compositions of the invention can contain synthetic or naturallecithin, or mixtures thereof. Natural preparations are used in somecases because they exhibit desirable physical characteristics, and/ormay be economical or nontoxic. However, in other embodiments,non-natural preparations are used, or the composition can include bothnatural and non-natural preparations.

Any suitable amount of phosphatidylcholine or lecithin may be presentwithin the composition. For example, at least about 0.25 wt %, at leastabout 0.5 wt %, at least about 1 wt %, at least about 2 wt %, at leastabout 3 wt %, at least about 5 wt %, at least about 8 wt %, at leastabout 10 wt %, at least about 20 wt %, at least about 30 wt %, at leastabout 40 wt %, at least about 50 wt %, at least about 60 wt %, at leastabout 70 wt %, at least about 80 wt %, or at least about 90 wt % of theentire composition can be a phosphatidylcholine or a lecithin. In somecases, the phosphatidylcholine or lecithin may be present at aconcentration of no more than about 95 wt %, no more than about 90 wt %,no more than about 80 wt %, no more than about 70 wt %, no more thanabout 65 wt %, no more than about 60 wt %, no more than about 50 wt %,no more than about 40 wt %, no more than about 30 wt %, no more thanabout 20 wt %, or no more than about 10%. Combinations of any of theseare also possible. For instance, the phosphatidylcholine or lecithin maybe present at between about 8 wt % and about 65 wt %, or between about 0wt % and about 10 wt %, etc. One or more than one type ofphosphatidylcholine or lecithin may be present.

Some delivery compositions of the present invention may containpolyenylphosphatidylcholine (herein abbreviated “PPC”). In some cases,PPC can be used to enhance epidermal penetration. The term“polyenylphosphatidylcholine,” as used herein, means anyphosphatidylcholine bearing two fatty acid moieties, wherein at leastone of the two fatty acids is an unsaturated fatty acid with at leasttwo double bonds in its structure, such as linoleic acid.

Certain types of soybean lecithin and soybean fractions, for example,can contain higher levels of polyenylphosphatidylcholine, withdilinoleoylphosphatidylcholine (18:2-18:2 phosphatidylcholine) as themost abundant phosphatidylcholine species therein, than conventionalfood grade lecithin. Such lecithins may be useful in formulating certaindelivery compositions. In some embodiments, conventional soybeanlecithin may be enriched with polyenylphosphatidylcholine, for instance,by adding soybean extracts containing high levels ofpolyenylphosphatidylcholine. As used herein, this type ofphosphatidylcholine is called “polyenylphosphatidylcholine-enriched”phosphatidylcholine (hereinafter referred to as PPC-enrichedphosphatidylcholine), even where the term encompasses lecithin obtainedfrom natural sources exhibiting polyenylphosphatidylcholine levelshigher than ordinary soybean varieties. These products are commerciallyavailable, for example, from American Lecithin Company, Rhone-Poulencand other lecithin vendors. American Lecithin Company markets itsproducts with a “U” designation, indicating high levels of unsaturation;Rhone-Poulenc's product is a soybean extract containing about 42%dilinoleoylphosphatidylcholine and about 24%palmitoyllinoleylphosphatidylcholine (16:0 to 18:2 of PC) as the majorphosphatidylcholine components. Another example of a suitablepolyenylphosphatidylcholine is NAT 8729 (also commercially availablefrom vendors such as Rhone-Poulenc and American Lecithin Company).

Any suitable amount of polyenylphosphatidylcholine may be present withinthe composition. For example, at least about 0.25 wt %, at least about0.5 wt %, at least about 1 wt %, at least about 2 wt %, at least about 3wt %, at least about 5 wt %, at least about 8 wt %, at least about 10 wt%, at least about 20 wt %, at least about 30 wt %, at least about 40 wt%, at least about 50 wt %, at least about 60 wt %, at least about 70 wt%, at least about 80 wt %, or at least about 90 wt % of the compositioncan be polyenylphosphatidylcholine. In some cases, thepolyenylphosphatidylcholine may be present at a concentration of no morethan about 95 wt %, no more than about 90 wt %, no more than about 80 wt%, no more than about 70 wt %, no more than about 65 wt %, no more thanabout 60 wt %, no more than about 50 wt %, no more than about 40 wt %,no more than about 30 wt %, no more than about 20 wt %, or no more thanabout 10%. Combinations of any of these are also possible. For instance,the polyenylphosphatidylcholine may be present at between about 8 wt %and about 65 wt %. In some embodiments, at least about 20 wt %, at leastabout 30 wt %, at least about 40 wt %, at least about 50 wt %, at leastabout 60 wt %, at least about 70 wt %, at least about 80 wt %, at leastabout 90 wt %, or about 100 wt % of all of the phosphatidylcholine orlecithin in the composition is polyenylphosphatidylcholine.

While not wishing to be bound to any theory, it is believed that thePPC-enriched phosphatidylcholine forms a bilayer enveloping nitric oxide(and in some embodiments, other adjunct ingredients, if present) tocreate the composition. The PPC-enriched phosphatidylcholine is believedto contribute to the stability of the nitric oxide, for example, byshielding the nitric oxide from water, and/or by enhancing itspenetration into the skin or other area.

The first phase also comprises, in some embodiments of the invention, afatty acid ester. Non-limiting examples include ascorbate palmitate orisopropyl palmitate. In some cases, the fatty acid ester is used as apreservative or an antioxidant. The composition can include any suitableamount of fatty acid ester, for example, at least about 1 wt %, at leastabout 3 wt %, at least about 5 wt %, at least about 10 wt %, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, at leastabout 50 wt %, etc. In some cases, no more than about 60 wt %, no morethan about 50 wt %, no more than about 40 wt %, no more than about 30 wt%, no more than about 20 wt %, no more than about 18 wt %, no more thanabout 15 wt %, no more than about 12 wt %, or no more than about 10 wt %of the composition is fatty acid ester. Combinations of any of these arealso possible. For example, the composition may be between about 0 wt %and about 10 wt % fatty acid ester. The composition may include one ormore than one fatty acid ester.

In certain embodiments, a composition such as those described herein canbe formulated to include a second phase. Typically, the second phase issubstantially immiscible with the first phase comprisingphosphatidylcholine or lecithin. Two phases that are substantiallyimmiscible are able to form discrete phases when exposed to each otherat ambient conditions (e.g., 25° C. and 1 atm) for extended periods oftime (e.g., at least about a day). The phases can be separateidentifiable phases (e.g., one may float above the other), or in somecases, the phases are intermingled, e.g., as in an emulsion. Thestability of the discrete phases may be kinetic and/or thermodynamic innature, in various embodiments.

In one set of embodiments, the second phase may comprise an emulsifierwhich causes the first phase comprising phosphatidylcholine or lecithinto form a liquid crystal, and/or vesicles such as micelles or liposomes.Typically, in a liquid crystal phase, vesicular structures such asmicelles, liposomes, hexagonal phases, or lipid bilayers can be formed.In some cases, multilamellar structures may be present within the liquidcrystal phase, although in other cases, only unilamellar structures maybe present. For example, in certain cases, the PPC-enrichedphosphatidylcholine can be loosely arranged in a multilamellar fashion,with nitric oxide and optional adjunct ingredients being bonded orotherwise entrapped or contained within the lipid bilayers formedtherein. In some cases, the first phase (e.g., comprising PPC-enrichedphosphatidylcholine) and the second phase can form a structure such asis disclosed in U.S. Pat. No. 7,182,956 to Perricone, et al. This isbelieved (without wishing to be bound by any theory) to form a looselyarranged, yet stable, PPC-enriched phosphatidylcholine-drug complex thatmay allow penetration and delivery of nitric oxide and/or peptides andoptional adjunct ingredients to the skin, e.g., to the dermalvasculature.

In one set of embodiments, the second phase comprises an emulsifier. Theemulsifier, in one embodiment, is a substance that is able to stabilizean emulsion by increasing its kinetic stability. The emulsifier may alsobe chosen in some cases to be relatively inert or non-toxic relative tothe skin.

In some embodiments, the second phase may comprise a polyglycol. Thepolyglycol may include a polyhydric alcohol of a monomeric glycol suchas polyethylene glycol (PEG) and/or polypropylene glycol (PPG). Forexample, the PEG or PPG may be PEG or PPG 200, 300, 400, 600, 1,000,1,450, 3,350, 4,000, 6,000, 8,000, and 20,000, where the numberindicates the approximate average molecular weight of the PEG or PPG. Asis understood by those of ordinary skill in the art, a polyglycolcomposition often will comprise a range of molecular weights, althoughthe approximate average molecular weight is used to identify the typepolyglycol. More than one PEG and/or PPG can also be present in certaininstances.

The second phase may comprise a surfactant in some embodiments.Non-limiting examples of surfactants include a siloxylated polyethercomprising dimethyl, methyl(propylpolyethylene oxide propylene oxide,acetate) siloxane commercially available from vendors such as DowCorning (Dow Corning 190 surfactant). Other examples of materials thatcan be used as (or within) the second phase include, but are not limitedto, 1,2-propanediol, or silicone fluids containing low viscositypolydimethylsiloxane polymers, methylparaben (p-hydroxy benzoic acidmethyl ester) commercially available from vendors such as Dow Corning(Dow Corning 200 silicone fluid). Still other examples include varioussiloxane or silicone compounds, e.g., hexamethyldisiloxane,amodimethicone, phenyltrimethicone, etc.

Additionally, purified water may be added to the second phase in someembodiments, although in other cases, little or no water is present inthe second phase. For example, the first phase, the second phase, cancontain less than 10%, less than 5%, less than 2%, less than 1%, or lessthat 0.05% (e.g., wt %) of water relative to the weight of therespective phase or of the entire composition. In some cases, the secondphase may also comprise adjunct ingredients such as those describedherein.

The second phase may include any one, or more than one, of the materialsdescribed above. In addition, any suitable amount of second phase can beused in accordance with various embodiments of the invention. Forexample, the second phase may be present at at least about 10 wt %, atleast about 20 wt %, at least about 30 wt %, at least about 40 wt %, atleast about 50 wt %, at least about 60 wt %, at least about 70 wt %, atleast about 80 wt %, or at least about 90 wt % of the composition. Insome cases, the ratio of the first phase (e.g., comprisingphosphatidylcholine or lecithin) to the second phase can be at leastabout 1:3, at least about 1:2, at least about 1:1, at least about 2:1,at least about 3:1, or at least about 4:1, etc.

In certain embodiments, a composition such as those described herein,e.g., those containing a peptide, and in some aspects also containingnitric oxide, may comprise a polyglycol. The polyglycol may include apolyhydric alcohol of a monomeric glycol such as polyethylene glycol(PEG) and/or polypropylene glycol (PPG). For example, the PEG or PPG maybe PEG or PPG 200, 300, 400, 600, 1,000, 1,450, 3,350, 4,000, 6,000,8,000, and 20,000, where the number indicates the approximate averagemolecular weight of the PEG or PPG. As is understood by those ofordinary skill in the art, a polyglycol composition often will comprisea range of molecular weights, although the approximate average molecularweight is used to identify the type polyglycol.

More than one PEG and/or PPG can also be present in certain instances.The composition can include any suitable amount of polyglycol, forexample, at least about 1 wt %, at least about 3 wt %, at least about 5wt %, at least about 10 wt %, at least about 20 wt %, at least about 30wt %, at least about 40 wt %, at least about 50 wt %, etc. In somecases, no more than about 60 wt %, no more than about 50 wt %, no morethan about 40 wt %, no more than about 30 wt %, no more than about 20 wt%, no more than about 18 wt %, no more than about 15 wt %, no more thanabout 12 wt %, or no more than about 10 wt % of the composition ispolyglycol. Combinations of any of these are also possible. For example,the composition may be between about 0 wt % and about 10 wt %polyglycol. The composition may include one or more than one type ofpolyglycol.

In some aspects, the formulation comprises a phosphatidylcholine, e.g.,any of those described herein (Phospholipon-90G (American LecithinCompany)). The composition can include any suitable amount ofphosphatidylcholine, for example, at least about 1 wt %, at least about3 wt %, at least about 5 wt %, at least about 10 wt %, at least about 20wt %, at least about 30 wt %, at least about 40 wt %, at least about 50wt %, at least about 60 wt %, at least about 70 wt %, at least about 80wt %, at least about 90 wt % etc. In some cases, no more than about 90wt %, no more than about 80 wt %, no more than about 70 wt %, no morethan about 60 wt %, no more than about 50 wt %, no more than about 40 wt%, no more than about 30 wt %, no more than about 20 wt %, no more thanabout 10 wt %, or no more than about 5 wt % of the composition isphosphatidylcholine. Combinations of any of these are also possible. Forexample, the composition may be between about 0 wt % and about 10 wt %surfactant. The composition may include one or more than onephosphatidylcholine. Other examples of materials that can be used as (orwithin) the formulation include, but are not limited to, benzyl alcohol,ethyl alcohol, isopropyl palmitate (IPP), propanediol, andcaprylic/capric triglycerides.

Any suitable amount of peptide may be present within a compositionprepared as described herein. One or more than one peptide may bepresent in some cases. For example, a peptide may be present at aconcentration, with respect to the volume of carrier (in cubiccentimeters) containing the peptide, of at least about 0.01 mg/cc, atleast about 0.02 mg/cc, at least about 0.03 mg/cc, at least about 0.04mg/cc, at least about 0.05 mc/cc, at least about 0.06 mg/cc, at leastabout 0.07 mg/cc, at least about 0.08 mg/cc, at least about 0.09 mg/cc,at least about 0.10 mg/cc, at least about 0.20 mg/cc, at least about0.30 mg/cc, at least about 0.40 mg/cc, at least about 0.50 mg/cc, atleast about 0.60 mg/cc, at least about 0.70 mg/cc, at least about 0.80mg/cc, at least about 0.90 mg/cc, at least about 1.0 mg/cc, at leastabout 1.5 mg/cc, at least about 2.0 mg/cc, at least about 2.5 mg/cc, atleast about 3.0 mg/cc, at least about 3.5 mg/cc, at least about 4.0mg/cc, at least about 4.5 mg/cc, at least about 5.0 mg/cc, at leastabout 5.5 mg/cc, at least about 6.0 mg/cc, at least about 6.5 mg/cc, atleast about 7.0 mg/cc, at least about 7.5 mg/cc, at least about 8.0mg/cc, at least about 8.5 mg/cc, at least about 9.0 mg/cc, at leastabout 9.5 mg/cc, at least about 10.0 mg/cc, at least about 15.0 mg/cc,at least about 20.0 mg/cc, at least about 25.0 mg/cc, at least about30.0 mg/cc, at least about 35.0 mg/cc, at least about 40.0 mg/cc, atleast about 45.0 mg/cc, at least about 50.0 mg/cc, at least about 60.0mg/cc, at least about 70.0 mg/cc, at least about 80.0 mg/cc, at leastabout 90.0 mg/cc, at least about 100 mg/cc, at least about 125 mg/cc, atleast about 150 mg/cc, at least about 175 mg/cc, at least about 200mg/cc, at least about 250 mg/cc, at least about 300 mg/cc, at leastabout 400 mg/cc, at least about 500 mg/cc, at least about 750 mg/cc, orat least about 1000 mg/cc.

In certain cases, the peptide may be present at a concentration of nomore than about 1500 mg/cc, no more than about 1000 mg/cc, no more thanabout 800 mg/cc, no more than about 600 mg/cc, no more than about 400mg/cc, no more than about 200 mg/cc, no more than about 100 mg/cc, nomore than about 75 mg/cc, no more than about 50 mg/cc, no more thanabout 25 mg/cc, no more than about 20 mg/cc, no more than about 15mg/cc, no more than about 12.5 mg/cc, no more than about 10.0 mg/cc, nomore than about 9.0 mg/cc, no more than about 8.0 mg/cc, no more thanabout 7.0 mg/cc, no more than about 6.0 mg/cc, no more than about 5.5mg/cc, no more than about 5.0, no more than about 4.5 mg/cc, no morethan about 4.0 mg/cc, no more than about 3.5 mg/cc, no more than about3.0 mg/cc, no more than about 2.5 mg/cc, no more than about 2.0 mg/cc,no more than about 1.5 mg/cc, no more than about 1.0 mg/cc, no more thanabout 0.5 mg/cc, no more than about 0.1 mg/cc, or no more than about0.01 mg/cc. Combinations of any of these are also possible. For example,the peptide may be present at a concentration of between about 0.5 mg/ccand about 50.0 mg/cc. In some embodiments, the composition alsocomprises a suitable amount of nitric oxide, e.g., as provided herein.

In some embodiments, a peptide may be added to a composition during orafter the formulation of any phase or composition as described herein,e.g., by routine methods known in the art. For example, the peptide maybe added to any phase of a formulation or composition, or after anyformulation or composition described herein is made. In some cases, forexample, the peptide may be added before or after nitric oxide is addedto the mixture, or before or after the first and second phases are mixedtogether. The peptide may be added to either the first phase or thesecond phase, depending on the type of peptide. In some cases, thepeptide may be added after the first phase or the second phase afteremulsification. The peptide may be present in a hydrophilic portion(e.g., a first portion) or a hydrophobic portion (e.g., a secondportion) of a formulation, depending on the peptide. For example, if theformulation is an emulsion, a hydrophilic peptide may be containedwithin a hydrophilic phase of the emulsion, while a hydrophobic peptidemay be contained within a hydrophobic phase of the emulsion; a peptidehaving both hydrophilic portions and hydrophobic portions may becontained at an interface between hydrophilic and hydrophobic phases ofthe emulsion. Accordingly, a variety of peptides, having differentproperties, may be used in various formulations and embodiments of thepresent invention, including any of the peptides described herein.Similarly, nitric oxide may be added during or after the formulation ofany phase or composition as described herein, e.g., by routine methodsand those described herein, e.g., by bubbling nitric oxide gas throughany phase, formulation, or composition, as described herein.

In another set of embodiments, the composition may also include one ormore transdermal penetration enhancers. Examples of transdermalpenetration enhancers include, but are not limited to,1,3-dimethyl-2-imidazolidinone or 1,2-propanediol. Other examplesinclude cationic, anionic, or nonionic surfactants (e.g., sodium dodecylsulfate, polyoxamers, etc.); fatty acids and alcohols (e.g., ethanol,oleic acid, lauric acid, liposomes, etc.); anticholinergic agents (e.g.,benzilonium bromide, oxyphenonium bromide); alkanones (e.g., n-heptane);amides (e.g., urea, N,N-dimethyl-m-toluamide); organic acids (e.g.,citric acid); sulfoxides (e.g., dimethylsulfoxide); terpenes (e.g.,cyclohexene); ureas; sugars; carbohydrates or other agents. Thetransdermal penetration enhancers can be present in any suitable amountwithin the composition. For example, at least about 10 wt %, at leastabout 20 wt %, at least about 30 wt %, at least about 40 wt %, or atleast about 50 wt % of the composition may comprise one or moretransdermal penetration enhancers. In some cases, no more than about 60wt %, no more than about 50 wt %, no more than about 40 wt %, no morethan about 30 wt %, no more than about 20 wt %, no more than about 10 wt%, no more than about 9 wt %, or no more than about 5 wt % of thecomposition comprises transdermal penetration enhancers. Combinations ofany of these are also possible. For example, the composition may havebetween about 0 wt % and about 5 wt % of one or more transdermalpenetration enhancers.

In other embodiments, the composition may be modified in order tocontrol depth of penetration. For example, in certain embodiments, thecomposition includes one or more polymers that act to reduce penetrationdepth of nitric oxide. Controlled depth of penetration may be importantfor indications where local administration is desired without systemiceffects. Examples of transdermal penetration barrier polymers include,but are not limited to, silicone waxes, acrylate polymers, anddimethicone copolymers. In certain embodiments, a transdermalpenetration barrier polymer is nonionic. A transdermal penetrationbarrier polymer can be present in any suitable amount within thecomposition. For example, at least about 10 wt %, at least about 20 wt%, at least about 30 wt %, at least about 40 wt %, or at least about 50wt % of the composition may comprise one or more transdermal penetrationbarrier polymers. In some cases, no more than about 60 wt %, no morethan about 50 wt %, no more than about 40 wt %, no more than about 30 wt%, no more than about 20 wt %, no more than about 10 wt %, no more thanabout 9 wt %, or no more than about 5 wt % of the composition comprisesa transdermal penetration barrier polymer. Combinations of any of theseare also possible. For example, the composition may have between about 0wt % and about 5 wt % of one or more transdermal penetration barrierpolymers.

As a specific non-limiting example of one set of embodiments, apolyenylphosphatidylcholine comprises a certain material with the tradename NAT 8729, and optionally at least one polyglycol (polyhydricalcohol of a monomeric glycol such as polyethylene glycol 200, 300, 400,600, 1,000, 1,450, 3,350, 4,000, 6,000, 8,000 and 20,000). Thecomposition can also comprise a PPC-enriched phosphatidylcholinematerial that is present within the first or second phase, e.g.,comprising nitric oxide, peptide, or both. The second phase may alsocomprise a surfactant such as a siloxylated polyether comprisingdimethyl, methyl(propylpolyethylene oxide propylene oxide, acetate)siloxane commercially available from vendors such as Dow Corning (DowCorning 190 surfactant) and lubricant such as silicone fluids containinglow viscosity polydimethylsiloxane polymers, methylparaben (p-hydroxybenzoic acid methyl ester) commercially available from vendors such asDow Corning (Dow Corning 200 silicone fluid).

In some embodiments, various compositions of the invention areformulated to be substantially clear or substantially transparent.Transparency may be useful, for instance, for product acceptance in themarketplace, e.g., when applied to the skin of a subject. However, inother embodiments, the composition is not necessarily transparent.Certain substances can be useful in providing a substantiallytransparent composition, for example, fatty acid esters such asascorbate palmitate or isopropyl palmitate. In one set of embodiments,the composition may be substantially transparent such that incidentvisible light (e.g., have wavelengths of between about 400 nm and about700 nm) can be transmitted through 1 cm of the composition with a lossin intensity of no more than about 50%, about 60%, about 70%, about 80%,or about 90% relative to the incident light. In some embodiments, theremay be no substantial difference in the wavelengths that are absorbed bythe composition (i.e., white light passing through the compositionappears white), although in other cases, there can be more absorption atvarious wavelengths (for example, such that white light passing throughthe composition may appear colored).

Other components may also be present within the composition, inaccordance with certain embodiments of the invention. For example, thecomposition may include volatile organic fluids, fatty acids, volatilearomatic cyclic compounds, high molecular weight hydrocarbons, or thelike.

In accordance with certain aspects of the invention, the composition maybe prepared by mixing a first phase and a second phase together, thenpassing nitric oxide through the mixture. As discussed above, the secondphase can comprise an emulsifier, or any other components discussedherein. The first phase may comprise a lecithin such asphosphatidylcholine and/or polyenylphosphatidylcholine, e.g.,PPC-enriched phosphatidylcholine, for instance, as described herein. Insome embodiments, other components are also mixed into the composition,before or after (or while) adding nitric oxide, for example, peptides,transdermal penetration enhancers, adjuvants, polyglycols (e.g., PEGand/or PPG), surfactants, lubricants, etc. as discussed herein. In someembodiments, however, nitric oxide may be passed through the first phaseprior to mixing of the first phase with the second phase.

In one set of embodiments, after forming the mixture, nitric oxide canbe passed into or through the mixture, for example, by blowing bubblesof nitric oxide through the mixture. Nitric oxide may be delivered intothe mixture under pressures such as between about 3,000 Pa and about15,000 Pa, between about 5,000 Pa and about 10,000 Pa, or between about6,000 Pa and about 8,000 Pa, and/or temperatures such as between about0° C. and about 50° C., between about 20° C. and about 35° C., or about25° C. and about 30° C. However, higher or lower pressures also may beused in some embodiments as aspects of the invention are not limited inthis respect.

In certain embodiments, the nitric oxide is bubbled through the mixtureuntil the mixture begins to at least partially solidify. As an example,the viscosity of the mixture may increase to at least about 1,000 cP, atleast about 2,000 cP, at least about 3,000 cP, at least about 5,000 cP,at least about 7,000 cP, at least about 10,000 cP, at least about 12,000cP, at least about 15,000 cP, at least about 20,000 cP, at least about30,000 cP, at least about 40,000 cP, at least about 50,000 cP, at leastabout 60,000 cP, at least about 70,000 cP, or at least about 80,000 cP.The nitric oxide can be passed through the mixture as pure nitric oxide,and/or with other gases (e.g., a noble gas, for example, argon). In somecases, a nitric oxide donor may be passed into the mixture, and therein,at least some of the nitric oxide donor can be converted into nitricoxide. In other embodiments, however, the final composition may havelower viscosities, for example, such that the composition is liquid, orcould be sprayed onto the skin.

In one set of embodiments, the nitric oxide can be bubbled through themixture to cause the viscosity of the mixture to increase. For example,the viscosity can increase until the mixture begins to form a gel, acream, a lotion, an ointment, a solid “stick,” or the like. A cream maybe, for example, a semi-solid emulsion, e.g., comprising a first phaseand a second phase. The first phase may be discontinuous (e.g.,comprising small droplets or vesicles, such as is discussed herein) andthe second phase may be continuous, or vice versa. In some cases,however, both the first phase and the second phase are co-continuouswithin the mixture.

In some embodiments of the invention, a composition may be prepared asdiscussed above, then diluted, e.g., with a diluent, to produce a finalcomposition. For example, a “stock” composition may be initiallyprepared, e.g., having a relatively high nitric oxide and/or peptideconcentration, then the stock composition diluted to produce a finalcomposition, e.g., before use, before storage, before packaging, etc. Insome embodiments, the diluent used may be a component as discussedherein (for example, forming at least a portion of the second phase),and the same or different materials than may be present in the initialcomposition may be used. The dilution ratio (amount of diluent added,relative to the initial composition) may be at least about 2, at leastabout 3, at least about 5, at least about 10, at least about 15, atleast about 20, at least about 25, at least about 30, at least about 50,or at least about 100, or any other suitable factor.

A composition may be prepared and/or stored at any suitable temperatureand under any suitable conditions. In some embodiments, for instance, acomposition can be prepared and/or stored under limited or no oxygenconditions, as oxygen can adversely react with nitric oxide. Thecomposition can also be prepared and/or stored under limited or nonitrogen and/or carbon dioxide, as both can also react adversely withnitric oxide. For instance, the composition may be prepared and/orstored in a sealed environment (e.g., stored in a sealed container). Thesealed environment (e.g., container) can be at least substantiallydevoid of gas, and/or contains a gaseous mixture that excludes, or atleast is depleted in, oxygen. In some embodiments, an environmentdepleted in oxygen may have less than about 20%, less than about 15%,less than about 10%, less than about 5%, about 1% or less, about 0.1% orless, about 0.01% or less, about 0.001% or less, oxygen (e.g., as a wt %or as molar % per volume). For example, the gaseous mixture may includea noble gas, such as argon, helium, neon, etc. In one set ofembodiments, the container may comprise a multi-layered metallic and/orpolymeric barrier, e.g., formed from Glaminate® (American Can Company).For instance, the container may have the shape of a tube. Thus, incertain embodiments, the container is substantially resistant to oxygenpermeation, nitrogen permeation, and/or carbon dioxide permeation. Incertain embodiments, the container is substantially watertight, forexample, such that substantially no water is absorbed by the container,or such that no water is able to pass through the container even if thecontainer is filled with water.

As previously discussed, nitric oxide can react with water, and thus,compositions described herein may be prepared and/or stored underconditions where substantially no water is present. For example, nitricoxide and/or a nitric oxide containing preparation described herein maybe prepared and/or stored under relatively low relative humidities(e.g., less than about 50% RH, less than about 40% RH, less than about30% RH, less than about 20% RH, or less than about 10% RH), and/or inthe presence of a suitable desiccant, such as phosphorous pentoxide orsilica gel.

In certain embodiments, the mixture may be mixed with or otherwiseinclude adjunct ingredients, if applicable, and nitric oxide may beintroduced to the mixture, e.g., bubbles of nitric oxide gas may beblown into the mixture until the mixture hardens to obtain the desiredfinal composition. As a specific non-limiting example, a nitric oxidecomposition may be formed by preparing a non-liposome multilamellarliquid crystal phosphatidylcholine phase, for example, by providing apolyglycol, then introducing phosphatidyl choline into the glycol atroom temperature to form a phosphatidylcholine solution. Thephosphatidylcholine often comes as a solid (e.g., as a “brick” ofmaterial), and the phosphatidylcholine may be broken down into smallerpieces to aid in mixing, e.g., by “shaving” or grinding thephosphatidylcholine solid. The phosphatidylcholine solution is mixeduntil the phosphatidylcholine solution is substantially clear, then onemay warm the phosphatidylcholine solution to 40° C., mill the warmedsolution (i.e., low agitation after the initial mixing), combinesiloxylated polyether and polydimethylsiloxane to form a fluid, add thefluid to the warmed solution and milling until the solution is clear,adding methyl paraben or other suitable lubricant to the solution andmilling until the methyl paraben dissolves in the solution, warm waterto 40° C. and adding the warmed water slowly to the solution, and thenceasing milling of the solution and “sweeping” the solution (e.g., witha sweep mixer) to cool to room temperature. Nitric oxide gas can then bebubbled or otherwise introduced into the solution while cooling thesolution until the solution begins to harden or becomes stiff, e.g.,having the consistency of a gel or a cream, such as previouslydescribed. In some cases, the resulting composition is sealed in acontainer, for example, as discussed herein. Any suitable container maybe used, e.g., a tube or a bottle. In addition, the composition (e.g.,within the container) may be stored at room temperature, or any othersuitable temperature. For example, a composition of the invention may bestored at or below 80° C., e.g., at or below room temperature (about 25°C.) or in a refrigerator (e.g., at 4° C.) for extended period ofstorage, for instance, to prevent nitric oxide leakage or denaturing. Insome cases, storage may extend for at least about a week, at least about4 weeks, at least about 6 months, at least about a year, etc.

It is surprising that, according to some embodiments, nitric oxide notonly can be entrapped in phosphatidylcholine or lecithin compositionssuch as those described herein, but also that such entrappedcompositions may have a long shelf life, especially when refrigerated.No loss or reaction of nitric oxide is expected during extendedrefrigerated storage, at least under certain conditions. For instance,in certain embodiments, the composition may be stored at temperatures ofless than about 80° C., less than about 70° C., less than about 60° C.,less than about 50° C., less than about 40° C., less than about 30° C.,less than about 25° C., less than about 20° C., less than about 15° C.,less than about 10° C., less than about 5° C., less than about 0° C.,etc., for extended periods of time, e.g., at least about a day, at leastabout a week, at least about 4 weeks, at least about 6 months, etc.

Without wishing to be bound by theory, it is believed that nitric oxideforms reversible physical bonds, similar to hydrogen bonds or van derWaals forces, with phosphatidylcholine or other lecithin molecules,e.g., containing one or more double bonds, which may allow nitric oxideto become entrapped and thereby remain intact for an extended period oftime, e.g., during storage. These physical bonds, however, are believedto be not very stable, and may in some cases be easily broken up, forexample, upon various physical agitations such as rubbing thecomposition against the skin, thereby releasing the entrapped nitricoxide. While others have stabilized other substances or drugs withinphosphatidylcholine or lecithin compositions or vesicles, for example,protein drugs such as insulin, it is surprising that a small, highlyreactive molecule such as NO could similarly be stabilized, especiallywhen it would have been expected that a molecule as small as NO wouldreadily diffuse away from such compositions and/or would have reactedwith water that is typically present within such compositions.

In some embodiments, it is believed that other species reactive withwater could also be similarly stabilized, e.g., within a composition asherein described. Any species that ordinarily reacts with water could bestabilized within such compositions. Examples of such species include,but are not limited to, lithium, or drugs or polymers with labile bondssusceptible to hydrolysis, for instance, certain peptides,polysaccharides, polylactic acid, polyglycolic acid, etc.

In certain aspects of the invention, a composition such as thosedescribed herein can be administered to a subject, such as a humansubject, by rubbing it on the skin of the subject, e.g., in areaslocated at or at least within the vicinity of a desired target area.Without wishing to be bound by any theory, it is believed thatphosphatidylcholine provides or facilitates delivery of nitric oxideand/or peptides to the skin, allowing nitric oxide and/or peptides to bedelivered to a target area. In some embodiments, the composition can beapplied, by rubbing the composition topically against the skin, whichallows the composition (or at least, nitric oxide and/or peptides) to beabsorbed by the skin. The composition can be applied once, or more thanonce. For example, the composition may be administered at predeterminedintervals. In some embodiments, for instance, the composition may beapplied once per day, twice per day, 3 times per day, 4 times per day,once every other day, once every three days, once every four days, etc.The amount of nitric oxide and/or peptides necessary to bring about thetherapeutic treatment is not fixed per se, and may depend upon factorssuch as the desired outcome, the type and severity the disease orcondition, the form of nitric oxide and/or peptides, the concentrationof nitric oxide and/or peptides present within the composition, etc.

Thus, another aspect of the invention provides methods of administeringany composition such as discussed herein to a subject. The compositionsof the invention may be applied to a subject for local or systemicdelivery, depending on the application. The compositions of theinvention may also be applied to any suitable area of the skin. Forexample, the compositions may be applied to the upper chest or arms, orto any hairy or non-hairy portion of the skin, e.g., to promote systemicdelivery of nitric oxide and/or peptides, or the composition may beapplied locally, e.g., at an area of the skin where treatment isdesired.

When administered, the compositions of the invention are applied in atherapeutically effective, pharmaceutically acceptable amount as apharmaceutically acceptable formulation. Any of the compositions of thepresent invention may be administered to the subject in atherapeutically effective dose. When administered to a subject,effective amounts will depend on the particular condition being treatedand the desired outcome. A therapeutically effective dose may bedetermined by those of ordinary skill in the art, for instance,employing factors such as those described herein and using no more thanroutine experimentation.

In some embodiments, an effective amount is an amount sufficient to havea measurable positive effect on blood flow and/or vasodilation, and/or ameasurable negative effect on blood pressure. In some embodiments, theeffect on blood flow and/or vasodilation is observed local to the siteof topical application. In some embodiments, an effective amount is anamount sufficient to have a measurable effect on cancer as evidenced byan appropriate clinical parameter, e.g., decrease in tumor size,decrease in cell proliferation, decrease in tumor biomarker, increase inprogression-free survival, increase in overall survival. In someembodiments, an effective amount is an amount sufficient to obtain asystemic level of nitric oxide and/or peptides that is sufficient tohave a desired effect, e.g., have a measurable positive effect on bloodflow and/or vasodilation, have a measurable negative effect on bloodpressure, and/or have a measurable effect on cancer as evidenced by anappropriate clinical parameter.

In one set of embodiments, compositions described herein can beadministered to a subject in a dosage range from between about 0.01 toabout 10,000 mg/kg body weight/day, about 0.01 to about 5000 mg/kg bodyweight/day, about 0.01 to about 3000 mg/kg body weight/day, about 0.01to about 1000 mg/kg body weight/day, about 0.01 to about 500 mg/kg bodyweight/day, about 0.01 to about 300 mg/kg body weight/day, about 0.01 toabout 100 mg/kg body weight/day.

In one set of embodiments, the dosage may be between about 0.01 mg andabout 500 g, between about 0.01 mg and about 300 g, between about 0.01mg and about 100 g, between about 0.01 mg and about 30 g, between about0.01 mg and about 10 g, between about 0.01 mg and about 3 g, betweenabout 0.01 mg and about 1 g, between about 0.01 mg and about 300 mg,between about 0.01 mg and about 100 mg, between about 0.01 mg and about30 mg, between about 0.01 mg and about 10 mg, between about 0.01 mg andabout 3 mg, between about 0.01 mg and about 1 mg, between about 0.01 mgand about 0.3 mg, or between about 0.01 mg and about 0.1 mg.

In another set of embodiments, the dosage may be at least about 0.01 mg,at least about 0.02 mg, at least about 0.03 mg, at least about mg, atleast about 0.05 mg, at least about 0.1 mg, at least about 0.2 mg, atleast about 0.3 mg, at least about 0.5 mg, at least about 1 mg, at leastabout 2 mg, at least about 3 mg, at least about 5 mg, at least about 10mg, at least about 20 mg, at least about 30 mg, at least about 50 mg, atleast about 100 mg, at least about 200 mg, at least about 300 mg, atleast about 500 mg, at least about 1 g, at least about 2 g, at leastabout 3 g, at least about 5 g, at least about 10 g, etc. In some cases,the dosage may be no more than about 10 g, no more than about 5 g, nomore than about 3 g, no more than about 2 g, no more than about 1 g, nomore than about 500 mg, no more than about 300 mg, no more than about200 mg, no more than about 100 mg, no more than about 50 mg, no morethan about 30 mg, no more than about 20 mg, no more than about 10 mg, nomore than about 5 mg, no more than about 3 mg, no more than about 2 mg,no more than about 1 mg, no more than about 0.5 mg, no more than about0.3 mg, no more than about 0.2 mg, no more than about 0.1 mg, no morethan about 0.05 mg, no more than about 0.03 mg, no more than about 0.02mg, no more than about 0.01 mg, etc. In some cases, combinations of anyof these are also possible, e.g., between about 0.01 mg and about 0.1mg.

The compositions described herein can be used in combination therapywith one or more additional therapeutic agents. For combinationtreatment with more than one active agent, where the active agents arein separate dosage formulations, the active agents may be administeredseparately or in conjunction. In addition, the administration of oneelement may be prior to, concurrent to, or subsequent to theadministration of the other agent. In certain embodiments, theadditional therapeutic agent is present in a provided composition inaddition to nitric oxide. In other embodiments, the additionaltherapeutic agent is administered separately from the nitric oxidecontaining composition.

When co-administered with other agents, an “effective amount” of thesecond agent will depend on the type of drug used. Suitable dosages areknown for approved agents and can be adjusted by the skilled artisanaccording to the condition of the subject, the type of condition(s)being treated and the amount of a compound described herein being used.In cases where no amount is expressly noted, an effective amount shouldbe assumed.

In certain embodiments, a nitric oxide and/or peptide containingcomposition as described herein, and the additional therapeutic agentare each administered in an effective amount (i.e., each in an amountwhich would be therapeutically effective if administered alone). Inother embodiments, a nitric oxide and/or peptide containing compositionas described herein, and the additional therapeutic agent are eachadministered in an amount which alone does not provide a therapeuticeffect (a sub-therapeutic dose). In yet other embodiments, a nitricoxide and/or peptide containing composition as described herein can beadministered in an effective amount, while the additional therapeuticagent is administered in a sub-therapeutic dose. In still otherembodiments, a nitric oxide and/or peptide containing composition asdescribed herein can be administered in a sub-therapeutic dose, whilethe additional therapeutic agent is administered in an effective amount.

As used herein, the terms “in combination” or “co-administration” can beused interchangeably to refer to the use of more than one therapy (e.g.,one or more prophylactic and/or therapeutic agents). The use of theterms does not restrict the order in which therapies (e.g., prophylacticand/or therapeutic agents) are administered to a subject.

Co-administration encompasses administration of the first and secondamounts of the compounds in an essentially simultaneous manner, such asin a single pharmaceutical composition, for example, capsule or tablethaving a fixed ratio of first and second amounts, or in multiple,separate capsules or tablets for each. In addition, suchco-administration also encompasses use of each compound in a sequentialmanner in either order. When co-administration involves the separateadministration of the first amount of a composition as described herein,and a second amount of an additional therapeutic agent, the compoundsare administered sufficiently close in time to have the desiredtherapeutic effect. For example, the period of time between eachadministration which can result in the desired therapeutic effect, canrange from minutes to hours and can be determined taking into accountthe properties of each compound. For example, a composition as describedherein, and the second therapeutic agent can be administered in anyorder within about 24 hours of each other, within about 16 hours of eachother, within about 8 hours of each other, within about 4 hours of eachother, within about 1 hour of each other or within about 30 minutes ofeach other.

More specifically, a first therapy (e.g., a prophylactic or therapeuticagent such as a composition described herein) can be administered priorto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second therapy to a subject.

Examples of therapeutic agents that may be combined with a compositionof this disclosure, either administered separately or in the samepharmaceutical composition, include, but are not limited to, alkylatingagents (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, ifosfamide), anti-metabolites (e.g.,azathioprine, mercaptopurine), vinca alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine), taxanes (e.g., paclitaxel),topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate, teniposide), cytotoxic antibiotics(e.g., actinomycin, anthracyclines (e.g., doxorubicin, daunorubicin,valrubicin, idarubicin, epirubicin), bleomycin, plicamycin, mitomycin),melphalan, busulfan, capecitabine, actinomycin, thalidomide, rituximab,fludarabine, mitoxantrone, methotrexate, cytarabine, procarbazine,lomustine, fluorouracil, oncovin, hydroxydaunorubicin, epirubicin, andbiologically active peptides (e.g., LH-RH agonists, somatostatins, cellpenetrating peptides).

Compositions described herein may also be used in combination withsurgery or radiation therapy.

In certain embodiments of the invention, the administration of variouscompositions of the invention may be designed so as to result insequential exposures to the composition over a certain time period, forexample, hours, days, weeks, months, or years. This may be accomplished,for example, by repeated administrations of a composition of theinvention by one or more of the methods described herein, or by asustained or controlled release delivery system in which the compositionis delivered over a prolonged period without repeated administrations.Administration of the composition using such a delivery system may be,for example, by a transdermal patch. Maintaining a substantiallyconstant concentration of the composition may be preferred in somecases.

In one set of embodiments, a composition such as is discussed herein maybe applied to the skin of a subject, e.g., at any suitable location. Insome embodiments, the suitable location is at a cancer site. In someembodiments, the suitable location is near a cancer site (e.g., within1-10, 10-30, 20-50 cm, etc.). In some embodiments, the suitable locationis at a site on the skin that is above the cancer, i.e., site of cancerdoes not necessarily have to be at the level of the skin. In someembodiments, the cancer can be below the skin, and the penetration depthof the formulation and/or amount added can be adjusted accordingly. Insome embodiments, the suitable location is an area that covers thecancer site and is, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 40, or 50 cm in diameter. In some embodiments, when thecomposition is administered for systemic delivery, the suitable locationcan be anywhere on the body. The composition may be contacted using anysuitable method. For example, the composition may be rubbed on, pouredon, applied with an applicator (e.g., a gauze pad, a swab, a bandage,etc.), or the like. In some cases, the composition can be a liquid, agel, a cream, a lotion, an ointment, a solid “stick,” or the like, thatcan be applied to the skin by hand, for example, by rubbing or spraying.

Compared to other means of administration, the use of topicaladministration in certain embodiments of the present invention hasvarious advantages, including one or more of the following. In somecases, administration of a composition and delivery of nitric oxideand/or peptides as discussed herein is easier and more effective thanother drug administration routes, for example, oral delivery. Unlikeoral administration where a substantial amount of nitric oxide and/orpeptides may be destroyed during the digestive process, nitric oxideand/or peptides delivered topically is not exposed to the digestivetract. Topical application may also allow, in some instances, relativelysteady delivery of nitric oxide and/or peptides to the desired targetarea without the cyclic dosages typical of orally or parenterallyadministered drugs. In some embodiments, topical application may alsoavoid toxic side effects associated with sustained increased levels ofnitric oxide and/or peptides typical of oral or parenteraladministration.

Compared to other topical delivery systems that employ nitric oxidedonors (an entity that is able to release nitric oxide, such asL-arginine, nitroglycerin, or amyl nitrite) as a nitric oxide source,various aspects of the present invention utilizing nitric oxide gas haveseveral advantages, including one or more of the following. Nitric oxidecan be released relatively quickly in some embodiments, because therelease does not necessarily involve chemical transformations of nitricoxide donors to release nitric oxide. The concentration of nitric oxidecan accumulate quickly upon topical administration, leading to goodtherapeutic effect in certain embodiments of the invention. In someembodiments, the release rate of nitric oxide can be controlled, forinstance, by physical actions (e.g., by controlling how much of thecomposition is applied to the skin), in comparison to nitric oxidedonors which release nitric oxide upon chemical stimulation. Moreover,certain embodiments of the present invention employ phosphatidylcholine,a component of cell membranes, as a carrier which improves thepenetration and absorption of nitric oxide into cells and tissues. Thus,certain compositions of the present invention will be non-toxic orbiocompatible.

The compositions of the present invention may additionally comprise oneor more adjunct ingredients, for instance, pharmaceutical drugs, skincare agents, and/or excipients. For example, compositions of theinvention may include additional ingredients such as salts, bufferingagents, diluents, excipients, chelating agents, fillers, drying agents,antioxidants, antimicrobials, preservatives, binding agents, bulkingagents, silicas, solubilizers, or stabilizers. Non-limiting examplesinclude species such as calcium carbonate, sodium carbonate, lactose,kaolin, calcium phosphate, or sodium phosphate; granulating anddisintegrating agents such as corn starch or algenic acid; bindingagents such as starch, gelatin or acacia; lubricating agents such asmagnesium stearate, stearic acid, or talc; time-delay materials such asglycerol monostearate or glycerol distearate; suspending agents such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone;dispersing or wetting agents such as lecithin or othernaturally-occurring phosphatides; thickening agents such as cetylalcohol or beeswax; buffering agents such as acetic acid and saltsthereof, citric acid and salts thereof, boric acid and salts thereof, orphosphoric acid and salts thereof; or preservatives such as benzalkoniumchloride, chlorobutanol, parabens, or thimerosal. Suitableconcentrations can be determined by those of ordinary skill in the art,using no more than routine experimentation. Those of ordinary skill inthe art will know of other suitable formulation ingredients, or will beable to ascertain such, using only routine experimentation.

Preparations can include sterile aqueous or nonaqueous solutions,suspensions and emulsions, which can be isotonic with the blood of thesubject in certain embodiments. Examples of nonaqueous solvents arepolypropylene glycol, polyethylene glycol, vegetable oil such as oliveoil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,organic esters such as ethyl oleate, or fixed oils including syntheticmono or di-glycerides. Aqueous solvents include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution,1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, antioxidants, chelatingagents and inert gases and the like. Those of skill in the art canreadily determine the various parameters for preparing and formulatingthe compositions of the invention without resort to undueexperimentation.

In one set of embodiments, the efficacy of various compositions of theinvention as applied to a subject may be determined or monitored bystudying the carotid arteries, which generally supply the head and neckwith blood. For example, in certain embodiments, a composition of theinvention may be applied to a subject for systemic delivery, and oneeffect of the nitric oxide may be to cause vasodilation. By studying thecarotid artery, the effect of the nitric oxide delivery may bemonitored, and if necessary, the dosing of nitric oxide adjusted. Anysuitable method may be used to monitor the carotid artery, e.g.,ultrasound, a carotid Doppler machine, functional MRI, PET scanning,etc. In other embodiments, if local delivery without systemic effects isdesired, the carotid artery can be monitored for any systemic effects.

In one aspect, one or more compositions described herein may beformulated for oral delivery. In some embodiments, one or morecompositions are provided in a capsule. A capsule can be a hard or softwater-soluble container, e.g., a gelatin container. Other examples ofcapsules include, but are not limited to polyglycolized glyceride,hydroxypropyl methylcellulose, Gelucire, iota carragennan, hydroxypropylstarch, polyvinyl alcohol, or the like, as well as combinations of theseand/or other materials. In some cases, other materials may be added tothe capsule shell, e.g., plasticizers, coloring agents, opacifiers, orthe like. Examples of plasticizers include glycerin or sorbitol. Acapsule can be coated to affect bioavailability and/or location ofrelease. A capsule can be used to target release to gastric, duodenal,intestinal, or colonic locations within the gastro-intestinal tract of asubject. Upon release a composition can adhere to the gastric mucosa anddeliver nitric oxide and/or peptide to the underlying tissue. In someembodiments, a composition described herein is mixed in a capsule withone or more fillers, diluents, glidants, or other agents. In someembodiments, a capsule wall is produced or coated to minimize oxygenand/or moisture penetration. In some embodiments, a composition is addedto a capsule under low oxygen and/or low humidity conditions. In someembodiments, a capsule is stored under low oxygen and/or low humidityconditions. Other capsule materials may be found in, e.g. Challenges andOpportunities in The Encapsulation of Liquid and Semi-Solid Formulationsinto Capsules for Oral Administration, Adv. Drug Deliv. Rev., 2008 Mar.17; 60(6):747-756, incorporated by reference herein in its entirety.

In another aspect, the present invention is directed to a kit includingone or more of the compositions discussed herein. A “kit,” as usedherein, typically defines a package or an assembly including one or moreof the compositions of the invention, and/or other compositionsassociated with the invention, for example, as described herein. Each ofthe compositions of the kit may be provided in liquid form (e.g., insolution), or in solid form (e.g., a dried powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species, which may or may not be provided with the kit.Examples of other compositions or components associated with theinvention include, but are not limited to, solvents, surfactants,diluents, salts, buffers, chelating agents, fillers, antioxidants,binding agents, bulking agents, preservatives, drying agents,antimicrobials, needles, syringes, packaging materials, tubes, bottles,flasks, beakers, dishes, frits, filters, rings, clamps, wraps, patches,containers, and the like, for example, for using, administering,modifying, assembling, storing, packaging, preparing, mixing, diluting,and/or preserving the compositions components for a particular use, forexample, to a sample and/or a subject.

A kit of the invention may, in some cases, include instructions in anyform that are provided in connection with the compositions of theinvention in such a manner that one of ordinary skill in the art wouldrecognize that the instructions are to be associated with thecompositions of the invention. For instance, the instructions mayinclude instructions for the use, modification, mixing, diluting,preserving, administering, assembly, storage, packaging, and/orpreparation of the composition and/or other compositions associated withthe kit. In some cases, the instructions may also include instructionsfor the delivery and/or administration of the compositions, for example,for a particular use, e.g., to a sample and/or a subject. Theinstructions may be provided in any form recognizable by one of ordinaryskill in the art as a suitable vehicle for containing such instructions,for example, written or published, verbal, audible (e.g., telephonic),digital, optical, visual (e.g., videotape, DVD, etc.) or electroniccommunications (including Internet or web-based communications),provided in any manner.

International Patent Application No. PCT/US2012/000151, filed Mar. 17,2012, entitled “Topical Nitric Oxide Systems and Methods of UseThereof,” by Nicholas V. Perricone, et al., is incorporated herein byreference in its entirety. In addition, the following applications, eachfiled on Sep. 19, 2012, each by Nicholas V. Perricone, are herebyincorporated by reference in their entireties: “Systems and Methods forTreatment of Acne Vulgaris and Other Conditions with a Topical NitricOxide Delivery System” (U.S. patent application Ser. No. 13/623,008);“Treatment of Skin and Soft Tissue Infection with Nitric Oxide” (U.S.patent application Ser. No. 13/623,010); “Methods and Systems forTreatment of Inflammatory Dermatoses with Nitric Oxide” (U.S. patentapplication Ser. No. 13/623,014); “Prevention and Treatment ofCardiovascular Diseases using Systems and Methods for Transdermal NitricOxide Delivery” (U.S. patent application Ser. No. 13/623,018);“Treatment and Prevention of Learning and Memory Disorders” (U.S. patentapplication Ser. No. 13/623,022); “Methods and Compositions for Muscularor Neuromuscular Diseases” (U.S. patent application Ser. No.13/622,998); “Compositions and Methods for Treatment of Osteoporosis andOther Indications” (U.S. patent application Ser. No. 13/623,004);“Techniques and Systems for Treatment of Neuropathic Pain and OtherIndications” (U.S. patent application Ser. No. 13/623,027); and “CancerTreatments and Compositions for Use Thereof” (U.S. patent applicationSer. No. 13/622,989).

Additionally, the following U.S. patent applications, each filed on Mar.13, 2013, are hereby incorporated by reference in their entireties:“Systems and Methods for Delivery of Peptides” (U.S. patent applicationSer. No. 13/801,402); “Treatment of Skin, Including Aging Skin, toImprove Appearance” (U.S. patent application Ser. No. 13/801,446); “HairTreatment Systems and Methods Using Peptides and Other Compositions”(U.S. patent application Ser. No. 13/801,488); “Skin Tanning UsingPeptides and Other Compositions” (U.S. patent application Ser. No.13/801,518); “Topical Systems and Methods for Treating SexualDysfunction” (U.S. patent application Ser. No. 13/801,543); “ImmuneModulation Using Peptides and Other Compositions” (U.S. patentapplication Ser. No. 13/800,952); “Cardiovascular Disease Treatment andPrevention” (U.S. patent application Ser. No. 13/801,013); “WoundHealing Using Topical Systems and Methods” (U.S. patent application Ser.No. 13/801,061); “Peptide Systems and Methods for Metabolic Conditions”(U.S. patent application Ser. No. 13/801,110); “Methods and Systems forTreating or Preventing Cancer” (U.S. patent application Ser. No.13/801,188); “Compositions and Methods for Affecting Mood States” (U.S.patent application Ser. No. 13/081,240); “Improvement of Memory orLearning Using Peptide and Other Compositions” (U.S. patent applicationSer. No. 13/801,298); and “Brain and Neural Treatments ComprisingPeptides and Other Compositions” (U.S. patent application Ser. No.13/801,345).

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

This example illustrates one technique for preparing a composition inaccordance with one embodiment of the invention. An accurate amount of acarrier (HNC 167-62) (see below) was introduced into a system. Thecarrier weight used in these experiments was approximately 250 g and thevessel size was 500 ml. The vessel was equipped with a mechanicalstirrer, gas inlet, and gas outlet and was previously purged with argonfor about an hour. The temperature of the carrier was kept at about25-30° C. NO gas regulated at 5 psi (1 psi is about 6,900 Pa) and wasthen introduced at a controlled rate of about 1 bubble/s with continuousstirring. The color, consistency, and viscosity of the carrier did notappear to change if NO was bubbled for 30 minutes to 2 hours. After 6hours, the weight of the carrier had increased by 0.15%, by 12 hours by0.25%, and by 24 hours by 0.56%. These increases in weight were believedto be significant considering the relative small molecular weight of NOversus the carrier. Although there was a slight change in color duringthe experiment (the color changed to slightly more orange), IR spectrumanalysis of the final product did not show any change versus the initialcarrier, indicating no noticeable chemical change in the carrier. Thecarrier also can solidify upon cooling if the carrier is initially asolid at lower temperature. Accordingly, this example demonstrates thata composition containing NO can be prepared in accordance with oneembodiment of the invention.

Example 2

In this example, six experiments were carried out to investigate theinteraction of nitric oxide with three carriers (HNC 157-62, HNC 157-65,and HNC 157-69) as well as with 1,3-propanediol, using experimentalconditions similar to that described for Example 1. In addition threeexperiments were performed to prepare carriers containing 800 ppm and500 ppm nitric oxide. HNC 157-62 was formed of 65% Phospholipon-90G(American Lecithin Company), 18% isopropyl palmitate (Kraft Chemicals),8% capric caprylic triglycerides (RITA Corp.), and 9% propanediol(Dupont). HNC 157-65 was formed of 65% Phospholipon-90G, 13% isopropylpalmitate, 14% capric caprylic triglycerides, 3% propanediol, and 5%dimethyl isosorbide (Croda). HNC 157-69 was formed from 65%Phospholipon-90G, 16% isopropyl palmitate, and 19% capric caprylictriglycerides.

The compositions were generally prepared as follows. Isopropylpalmitate, capric caprylic triglyceride, propanediol (for HNC 157-62 andHNC 157-65), and dimethyl isosorbide (for HNC 157-65) were mixedtogether and warmed to 40° C. Phospholipon-90G was then gradually addedto this liquid mixture by mixing it. Phospholipon-90G is typicallyreceived as individual pellets, and is mixed into the solution untilfully dissolved. The mixture was subsequently filtered through a sieveto remove any undissolved Phospholipon-90G.

Accordingly, the HNC carriers included 1,3-propanediol,Phospholipon-90G, isopropyl palmitate, capric and/or caporictriglycerides, and Arlasolve DMI (ICI America or Croda). Isopropylpalmitate, the capric and/or caporic triglycerides, and Arlasolve DMIare expected to be chemically inert towards nitric oxide, while theliterature suggests that 1,2-propanediol and glycerol may be able toreact with nitric oxide gas to form mononitrates. Accordingly, it wouldbe expected that 1,3-propanediol may also react with NO to formmononitrates:

In addition, Phospholipon-90G is derived from soybean and containsesters of unsaturated fatty acids such as oleic, linoleic, and linolenicacids, and thus, the unsaturated fatty acid part of Phospholipon-90Gwould react with nitric oxide to lead to a variety of nitrated products.

Each carrier was taken in a 500 mL three necked flask equipped with amechanical stirrer, gas inlet and a gas outlet. The system was purgedwith argon for one hour at room temperature (25° C.). Then nitric oxidegas was bubbled into the system. Then, nitric oxide gas was bubbledthrough carrier for stipulated amount of time. The changes in weight andcolor were noted. The details of individual experiments were as follows.

Experiment 1. The carrier was HNC 157-62. Nitric oxide gas was bubbledfor 24 hours at 25° C. The initial weight of carrier was 168.53 g., andthe final weight was 169.48 g. The net weight gained was 0.95 g and thepercentage weight gain was 0.56%.

Experiment 2. The carrier used was HNC 157-62. Nitric oxide gas wasbubbled for 48 hours at 25° C. The initial weight of carrier was 171.31g., and the final weight was 174.21 g. The net weight gained was 2.90 Gand the percentage weight gain was 1.69%.

Experiment 3. In order to differentiate between chemical reaction vs.physical absorption, the above reaction mixtures were heated at 55-60°C. for four hours. Minimal loss of weight was observed (˜200 mg),indicating no loss of absorbed nitric oxide gas. However, more intenseorange color developed during this process, indicating somedecomposition of the nitrites formed.

Experiment 4. The carrier used was HNC 157-65. Nitric oxide gas wasbubbled for 24 hours at 25° C. The initial weight of carrier was 171.66g., and the final weight was 172.98 g. The net weight gained was 1.32 gand percentage weight gain was 0.77%.

Experiment 5. The carrier used was HNC 157-69 (same as HNC 157-62,except it had no 1,3-propanediol). Nitric oxide gas was bubbled for 40hours at 25° C. The initial weight of carrier was 171.02 g., and thefinal weight was 171.97 g. The net weight gained was 0.95 g and thepercentage weight gain was 0.56%.

Experiment 6. Nitric oxide gas was bubbled through 1,3-propanediol(neat) for 40 hours at 25° C. The initial weight of the 1,3-propanediolwas 178.81 g., and the final weight was 178.97 g. The net weight gainedwas 0.16 g and the percentage weight gain was 0.09%.

Experiment 7. For preparation of 800 ppm NO, the carrier used was HNC157-62. Nitric oxide gas was bubbled for 2 hours at 25° C. The initialweight of carrier was 238.16 g., and the final weight was 238.35 g. Thenet weight gained was 0.19 g and the percentage weight gain 0.0798%(˜800 ppm). See entry 5 in Table 1.

Experiment 8. For preparation of 500 ppm NO, the carrier used was HNC157-65. Nitric oxide gas was bubbled for 2 hours at 25° C. The initialweight of carrier was 250.37 g., and the final weight was 250.50 g. Thenet weight gained was 0.13 g and the percentage weight gain was 0.0519%(˜500 ppm). See entry 6 in Table 1.

Experiment 9. For preparation of 800 ppm NO, the carrier used was HNC157-62. Nitric oxide gas was bubbled for 15 min at 25° C. The initialweight of carrier was 252.24 g., and the final weight was 252.45 g. Thenet weight gained was 0.21 g and the percentage weight gain 0.083% (˜800ppm).

These experiments were conducted with carriers the HNC 157-62, HNC157-65, HNC 157-69, and 1,3-propanediol.

As described above and in Table 1, weight gains ranging from 0.5% to1.7% were observed when nitric oxide gas was passed through thecarriers. In order to determine the nature of interaction between nitricoxide and carrier, the carrier was heated after nitric oxide absorptionat 60° C. for four hours. Practically no loss of weight was observed,which indicated that the nitric oxide gas reacted chemically with thecarriers (entries 1-4 in Table 1).

In order to investigate the reactivity of 1,3-propanediol with nitricoxide, nitric oxide absorption was studied using (a) HNC 157-69, whichdid not contain 1,3-propanediol, and (b) 1,3-propanediol by itself. HNC157-69 gained 0.95 g or 0.56% weight, much lower compared to its1,3-propanediol containing analog HNC 157-62, which showed 1.69% weightgain (entries 2 and 5 of Table 1). 1,3-propanediol itself, surprisingly,showed only negligible, if any, weight gain when NO was passed throughit (entry 6 in Table 1). Thus, under experimental conditions,1,3-propanediol did not react with nitric oxide.

Two samples were also prepared containing 800 ppm NO (from carrier HNC157-62) and one sample containing 500 ppm NO (from carrier HNC 157-65)(entries 7-9 in Table 1). The IR spectra of the carriers did not showany additional bands after the reaction, possibly because of low amountsof nitrites and/or overlap with the carrier complex bands.

Mass spectral studies of the carrier HNC 157-62 and HNC 157-62containing NO indicated that there was an increase in the intensity ofthe peak at m/e 104 in NO-containing carrier, compared to carrierwithout NO. The peak at m/e 104 was believed to be due to choline cation(C₅H₁₄NO). Phospholipon-90G may contain some free choline, and hencepresence of the peak at 104 in the mass spectrum of the carrier was notsurprising. However, the increase in the amount of choline after passageof NO was somewhat unexpected, although it is believed that nitric oxidecatalyzes similar dephosphorylation of Phospholipon-90G releasingcholine.

In conclusion, an increase in weight (0.56 to 1.69%) was observed whennitric oxide gas was passed through the carriers. 1,3-propanediol failedto gain any significant weight when nitric oxide was passed through it.HNC 157-69 (devoid of 1,3-propanediol) gained only 0.56% weight comparedto 1.69% by its 1,3-propanediol containing analog HNC 157-62. The massspectra of HNC 157-62 before and after passing NO indicated that thepeak corresponding to choline at m/e 104 increased after the passage ofNO, which suggests that phospholipon-90G may undergo NO-catalyzeddephosphorylation.

TABLE 1 Initial wt. Final wt. Time Temp. % Wt. Expt. No. Carrier g. g.hr ° C. Wt. Gain g gain 1 HNC 157- 168.53 169.48 24 25 0.95 0.56 62 2HNC 171.31 174.21 48 25 2.90 1.69 157-62 3 HNC 174.21* 174.01 4 60 −0.20−0.11 157-62 4 HNC 171.66 172.98 24 25 1.32 0.77 157-65 5 HNC 171.02171.97 40 25 0.95 0.56 157-69 6 1,3- 178.81 178.97 40 25 0.16 0.09Propanediol 7 HNC 238.16 238.35 2 25 0.19 0.0798 157-62 (~800 ppm) 8 HNC250.37 250.50 2 25 0.13 0.0519 157-65 (~500 ppm) 9 HNC 252.24 252.450.25 25 0.21 0.0833 157-62 (~800 ppm)

Example 3

This example illustrates non-invasive blood pressure measurements inmice using a composition in accordance with one embodiment of theinvention.

Blood pressure in mice may be measured using blood volume changes in themouse tail. Mice with normal tails (no clipping or short) were used inthis study. Ages varied between 8 weeks and 24 weeks. This procedureuses the CODA non-invasive blood pressure system available from KentScientific (Torrington, Conn.) Mice weighing approximately 25 grams wererestrained in plastic cylindrical housing with a nose come allowing thenose to protrude. Two tail cuffs provided occlusion and measurements.The O-cuff provided period occlusion while the VPR cuff providesvolume-pressure recordings. The occlusion pressure and the recordedpressure were controlled automatically by the computer software.

Each measurement had 10 acclimation cycles and 20 measurement cyclesonce daily depending on the experimental parameters. The average bloodpressure of male mice made over time was 136/88. In this study, bloodpressure measurements of the control (base) mice and the test (treated)mice were made and averaged over a period of three days. The testcomposition comprised 800 ppm nitric oxide, which was applied over theupper back of the animal in a quantity that exceeded 50 mg. The bloodpressures were recorded after each application and over 1 hour. Thefollowing results were obtained.

TABLE 2 Average Blood Pressure Reading Post Application Control (Base)Product Test (Treated) NO 800 ppm Time 0 167/123 140/91 1 Hour 170/128115/69This study shows that the application of the test product is capable ofreducing the blood pressure in a mouse to a significantly lower value.

Example 4

This example illustrates capillary blood flow measurements in humansusing a composition similar to the ones described in the above examples.

Microcirculation properties of the skin were measured before and afterapplication of a test product with nitric oxide. Measurements were madeusing a Moor® Laser Doppler instrument. Measurements were made at Day 1before and immediately, 5 and 15 minutes after treatment.

The study participants were healthy females aged 30 to 55 years. Theywere in good health as determined by the medical history and were nottaking any prescription medications. The test product was labeled as 800ppm NO (nitric oxide). The study participants had 100 mg of the testproduct applied in a 2×2 sq. inch area on the forearm.

Laser Doppler was performed to measure increased stimulation of themicro-capillary blood flow to skin. The micro-circulation of the skinreflects the perfusion of the skin and the underlying tissue. The laserDoppler technique is the standard method to obtain dynamic measurementof capillary blood flow in clinical evaluation. Measurements can be maderelatively rapidly and simultaneously at sites. In addition, temperaturemeasurements may also be made at the same time.

A Moor Instruments DRT4 Laser Doppler Blood Flow Monitor (Devon,England) was used. The laser Doppler technique measures blood flow inthe microcapillaries of the skin that are close to the skin surface andthe blood flow in underlying arterioles and venules that help toregulate skin temperature. There are several parameter used to describeblood flow measured by this laser Doppler technique. These measurementparameters are defined by Moor Instruments Inc. and are listed below.

Flux: This parameter is related to the product of average speed andconcentration of moving red blood cells in the tissue sample volume. Itis the parameter most widely reported in Laser Doppler publication.

Conc: This parameter gives an indication of the number of moving redblood cells in the tissue sample volume.

Speed: This parameter gives an indication of the average speed of redblood cells moving in the tissue sample volume.

Temp: This is the probe temperature and where there is good thermalconduction between probe and tissue it reaches tissue temperature.

Because of the nature of blood flow in the capillaries and other smallblood vessels, absolute flow units such as ml/minute cannot beexpressed. Therefore, arbitrary units are used. Blood flow changes aredefined as the percentage change from the baseline of these arbitraryunits.

The procedure was as follows. The laser Doppler probe was attached ontothe volar forearm. Control untreated skin readings (Baseline) wereobtained for 15 minutes. The test product was then applied in thedesignated 2×2 sq. inch area and rubbed into the skin. Readings wereobtained for 15 minutes.

The areas not used for evaluation include the first 15 seconds afterstarting data collection. Four 10 second areas in the baseline and testreadings at each time point were randomly selected to obtain the meanaverages which were then used in further analysis of the data.

The averaged data was compiled from the 4 study participants and thelaser Doppler results are provided in the FIGURE. There was asignificant difference observed in the control untreated skin and theskin treated with 800 ppm nitric oxide up to 15 minutes afterapplication. The applied nitric oxide had an effect on the microcirculation of the skin at the applied level (100 mg in a 2×2 sq. incharea). The topical test product is capable of passing through the skinand affecting the microcirculation of the skin.

Example 5

This example illustrates delivery of nitric oxide formulations similarto those discussed herein in humans. In these studies, laser Dopplerstudies were performed on three human female subjects. As discussedbelow, all of the studies showed positive results consistent with aphysiological effect of nitric oxide applied to the skin, passingthrough the skin and affecting the capillary circulation. Theformulations used produce positive, almost immediate results whenapplied to the skin which, in these studies, was manifested byvasodilatation of the cutaneous vascular system.

While nitric oxide has many physiological effects, the purpose of thesestudies was to measure physiological effects that would be relativelyeasy to determine, and which would be noninvasive. Laser Doppler wasselected for these studies because laser Doppler has a relatively largedatabase that indicates that it is effective in determining an increasein microcirculation, i.e., circulation within the capillary bedimmediately under the epidermal layer in the skin. Nitric oxide iscapable of violating the capillary bed, and thus, laser Doppler wasselected.

The formulation used in these studies contained nitric oxide dispersedin a lipid matrix. The nitric oxide was dispersed in the matrix and doesnot appear to be dissolved but remained intact, i.e., it appeared todiffuse into the skin as a molecule of nitric oxide rather than asatomic components or ions. Nitric oxide is a very rapid acting molecule,and these studies used a system that employed laser Doppler with acovered chamber. The formulation was placed into the chamber and thenattached to the skin by an adhesive layer on the covering. This provideda stable measuring device as determined by multiple normal evaluationsof the capillary blood flow without treatment of nitric oxide.

A known positive control, methyl nicotinate, was applied to the skin ata concentration of 1/10% in alcohol. There was a rapid response typicalof the vasodilator. The vascular dynamics are such that when the bloodvessels dilate physical parameters follow Bernoulli's law. This statesthat as one increases the diameter of a tube containing a liquid to flowwill increase but the pressure and the speed of the liquid willdecrease. The laser Doppler device accurately measured these parameters.

When nitric oxide was applied to the forearm of three human femalesubjects, it was observed that there was an immediate effect on theblood flow as soon as the formulation was applied. The speed of bloodflow decreased, and the effects lasted over 15 minutes. There was noerythema and no discomfort to the subjects that was observed.

Thus, these studies showed that the nitric oxide in the lipid matrixwhen applied topically to the forearm, was able to penetrate the skinvery rapidly and to interact with the underlying tissues. This isevidenced by the observation of vascular dilatation without erythema.Accordingly, it can be concluded from these studies that the formulationcontaining nitric oxide was effective in delivering nitric oxide throughthe skin in a physiologically active state.

In these studies, the participants were healthy females aged 30 to 55years. They were in good health as determined by the medical history andwere not taking any prescription medications. All study participantsread and signed the informed consent statement prior to any studyprocedures being performed.

The test product was labeled as 10,000 ppm NO (nitric oxide). The studyparticipants had 100 mg of the test product applied in a closed Hilltopchamber on the forearm.

Laser Doppler was performed to measure increased stimulation of themicro-capillary blood flow to skin. The micro-circulation of the skinreflected the perfusion of the skin and the underlying tissue. LaserDoppler is a standard method to obtain dynamic measurement of capillaryblood flow in clinical evaluation. Measurements can be made relativelyrapidly and simultaneously at sites. In addition, temperaturemeasurements may also be made at the same time.

A Moor Instruments DRT4 Laser Doppler Blood Flow Monitor (Devon,England) was used in these studies. The laser Doppler technique measuredblood flow in the microcapillaries of the skin that are close to theskin surface and the blood flow in underlying arterioles and venulesthat help to regulate skin temperature. Because of the nature of bloodflow in the capillaries and other small blood vessels, it is difficultto determine absolute flow units such as ml/minute. Therefore, arbitraryunits were used in these experiments to determine relative changes.Blood flow changes were accordingly defined as the percentage changefrom the baseline using the arbitrary units.

Data was compiled from the three study participants and the laserDoppler studies and averaged. It observed that there was a significantdifference observed in the control untreated skin and the skin treatedwith 10,000 ppm nitric oxide up to 15 minutes after application. Theapplied nitric oxide had an effect on the microcirculation of the skinat the applied level (100 mg in a lipid matrix). The formulation wasdetermined to be capable of passing through the skin and affecting themicrocirculation of the skin.

Example 6

This example illustrates a protocol used for determining the amount ofnitric oxide released from various compositions of the presentinvention. The protocol is generally performed as follows:

1) Samples (within HNC carriers described herein) were maintained ateither 4° C. or −20° C. until analyzed.

2) One at a time and before they were opened, samples were warmed to 37°C. (dry bath) in a controlled atmosphere of 1% O₂. They were opened and150 microliters was removed and placed into 1.35 mL of PBS (pH 7.4, 25°C. and equilibrated to 13 micromolar O₂) in a small glass screw-cap vialminimizing headspace.

3) The vials were then subjected to 15 s of vortex agitation, capsopened and 1 mL quickly removed and immediately injected into a SieversNitric Oxide Analyzer reaction chamber containing 4 mL of deionized anddistilled H₂O equilibrated at 0% O₂ by a N₂ flow through gas andmaintained at 37° C. by flow-through water jacket.

4) Peaks were analyzed by comparing values to a standard curve generatedby injecting various concentrations of the NO donor1-(hydroxy-NNO-azoxy)-L-proline (PROLI-NONOate). To standardize amongsamples, the area under the curve from 0 to 4 min was used forconcentration determinations are expressed in moles of NO and have beencorrected for the 2 dilutions (50×). As such, these values equal molesof NO/150 microliter matrix.

5) The authenticity of NO formation was validated by injecting 50 mM ofthe NO spin trap2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide(cPTIO) or by injecting sample into the reaction chamber containing 50mM cPTIO and observing the absence of signal.

6) For each sample, the area under the curve from t=0 to t=4 min wascalculated for concentration determinations. Calculations represent onlythe amount of NO released during this 4 min time span and thus do notrepresent the total amount of NO contained in the volume of matrixassessed (150 microliters). For each vial 3-5 independent determinationswere performed (this means each injection represents removal of 150microliters of matrix from the labeled vial, dilution in PBS andinjection. As such, these were completely independent measurements frombeginning to end.

TABLE 3 Sample ID Mean (millimoles NO) Std Dev 1000 11.4 1.55 4000 60.71.70 7000 72.1 11.9 10000 127.9 11.3In this table, the Sample ID numbers correspond to the amount of nitricoxide, in ppm, that was formulated in the composition (sample) tested.

Based on these results, the compositions tested here appeared to beeffective in entrapping nitric oxide gas, and are effective in releasingthe trapped gas in a measureable and significant way.

Example 7

This example illustrates techniques for preparing compositions inaccordance with one or more embodiments of the invention. Specifically,four formulations suitable as peptide and nitric oxide carriers areprovided: two four-phase formulations (HNC 156-43, HNC 156-50), and twosingle-phase formulations (HNC 156-47, HNC 159-136).

HNC 156-43

This formulation was formed of: 77.7% water, 6.0% Phospholipon-90G(American Lecithin Company), 0.10% EDTA-Na_(z) (Sigma), 0.1% citricacid, 5.0% isopropyl palmitate (IPP, Kraft Chemicals), 5.0% Promulgen-D(Lubrizol), 3.0% Arlacel-165 (Croda), 1.0% cetearyl alcohol 50/50, 0.5%Dow Corning Fluid 200-10 CST, 0.1% Tocotrienol-50C (Carotech), 0.5%Optiphen Plus (Lotioncrafter), and 1.0% Seppitonic M3 (Seppic).

The individual ingredients were divided between four phases as follows:

Phase 1: water, Phospholipon-90G, EDTA-Na_(z), citric acid.

Phase 2: IPP, Promulgen-D, Arlacel-165, cetearyl alcohol 50/50, DowCorning Fluid 200-10 CST, Tocotrienol-50C.

Phase 3: Optiphen Plus.

Phase 4: Seppitonic M3.

HNC 156-50

This formulation was formed of: 78.3% water, 10.0% Phospholipon-90G(American Lecithin Company), 0.10% EDTA-Na_(z) (Sigma), 0.2% sodiumhyaluronate (1% solution), 3.0% isopropyl palmitate (IPP, KraftChemicals), 3.0% Promulgen-D (Lubrizol), 4.0% Arlacel-165 (Croda), 0.7%cetearyl alcohol 50/50, 0.5% Optiphen Plus (Lotioncrafter), and 0.2%dimethylethanolamine (DMAE, Sigma).

The individual ingredients were divided between four phases as follows:Phase 1: water, Phospholipon-90G, EDTA-Na_(z), sodium hyaluronate.

Phase 2: IPP, Promulgen-D, Arlacel-165, cetearyl alcohol 50/50.

Phase 3: Optiphen Plus.

Phase 4: DMAE.

The four-phase compositions were generally prepared as follows: Phases 1and 2 were heated to 60° C., added and mixed together, then allowed tocool to 48° C., at which point Phase 3 was added and mixed. The mixturewas then allowed to cool to 38° C., at which point phase 4 was added andmixed.

HNC 156-47

This formulation was formed of: 47% water, 5.0% PEG-200 (Sigma), 45%Phospholipon-90G (American Lecithin Company), 1.0% benzyl alcohol, and2.0% ethyl alcohol.

HNC 159-136

This formulation was formed of: 65% Phospholipon-90G (American LecithinCompany), 18.0% isopropyl palmitate (IPP, Kraft Chemicals), 8% capriccaprylic triglycerides (RITA Corp.), and 9% propanediol (Dupont).

Any of the above compositions are effective as carriers for peptides andnitric oxide. For example, the TRH peptide was shown to be effectivelycarried by each of the above formulations at a TRH concentration of 5.0mg/cc.

Example 8

This example illustrates delivery of nitric oxide formulations to fivehuman subjects, using procedures and formulations similar to thosediscussed in Example 5. Each person served as their own control for thetesting. A Moor Instruments Laser Doppler was used to determinecirculation. All of these studies showed positive results consistentwith a physiological effect of nitric oxide applied to the skin, passingthrough the skin and affecting the capillary circulation. Theformulations used produce positive, almost immediate results whenapplied to the skin which, in these studies, was manifested byvasodilatation of the cutaneous vascular system.

For each subject, the following creams were applied: a negative controlformulation (containing no NO), 0.010 gram of a 10,000 ppm NOformulation, 0.020 gram of a 10,000 ppm NO formulation, and a positivecontrol formulation containing 0.1% methyl nicotinate. The formulationswere prepared using procedures similar to those discussed in Example 1.

When nitric oxide was applied to the forearm of the subjects, it wasobserved that there was an immediate effect on the blood flow as soon asthe formulation was applied. The speed of blood flow decreased, and theeffects lasted over 15 minutes. There was no erythema and no discomfortto the subjects that was observed.

Data was compiled from the study participants and the laser Dopplerstudies and averaged. It observed that there was a significantdifference observed in the control untreated skin (where no detectablevasodilation was observed), and the treatments involving 0.010 g of NO,0.020 g of NO, and methyl nicotinate. For all three treatments,vasodilatation was observed, with somewhat greater vasodilatation forthe 0.010 g formulation over the 0.02 g formulation. In addition, theamount of vasodilatation observed for the 0.01 g formulation of NO wasgenerally similar to the amount of vasodilatation observed for methylnicotinate.

Accordingly, these data demonstrate that NO as applied in a cream to theskin is able to penetrate the skin and affect bloodflow beneath theskin.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method, comprising: administering, to the skinof a subject having or at risk of cancer, a composition comprising aneffective amount of thyrotropin-releasing hormone (TRH) and molecularnitric oxide to treat or prevent the cancer, and a carrier having aphosphatidylcholine component entrapping the nitric oxide, wherein thecomposition is stable at room temperature.
 2. The method of claim 1,wherein the composition further comprises melanocyte-stimulating hormone(MSH).
 3. The method of claim 1, wherein the nitric oxide is molecularnitric oxide.
 4. The method of claim 3, wherein the molecular nitricoxide is present within the carrier as a gas or bound by hydrogen bondsor van der Waals forces.
 5. The method of claim 1, wherein thephosphatidylcholine carrier stabilizes the nitric oxide at a temperatureat or lower than 80° C.
 6. The method of claim 1, wherein thecomposition comprises a liquid crystal structure comprising thephosphatidylcholine.
 7. The method of claim 6, wherein at least aportion of the liquid crystal structure is multilamellar.
 8. A method,comprising contacting the skin of a subject having or at risk of cancerwith a composition comprising: an emulsion comprising a first phasecomprising thyrotropin-releasing hormone (TRH), molecular nitric oxide,and lecithin, and a second phase comprising an emulsifier, wherein thelecithin is present at least about 0.25% by weight of the composition,and wherein the first phase comprises no more than about 250 ppm ofwater by weight of the composition.
 9. The method of claim 8, whereinthe composition further comprises melanocyte-stimulating hormone (MSH).10. The method of claim 8, wherein the nitric oxide is molecular nitricoxide.
 11. The method of claim 10, wherein the molecular nitric oxide ispresent within the emulsion as a gas or bound by hydrogen bonds or vander Waals forces to the lecithin.
 12. The method of claim 8, wherein thefirst phase forms discrete vesicles contained within the second phase.13. The method of claim 8, wherein the nitric oxide is present at leastabout 0.5% by weight of the composition without nitric oxide.
 14. Themethod of claim 8, wherein at least some of the nitric oxide is presentwithin the first phase bound to phosphatidylcholine.
 15. The method ofclaim 8, wherein the lecithin comprises a phosphatidylcholine.
 16. Themethod of claim 15, wherein at least some of the phosphatidylcholinecomprises a polyenylphosphatidylcholine.
 17. The method of claim 8,wherein the first phase comprises a fatty acid ester.
 18. A method,comprising: administering, topically to the skin of a subject, acomposition comprising an emulsion comprising a first phase comprisingan effective amount of thyrotropin-releasing hormone (TRH), molecularnitric oxide, and phosphatidylcholine to a subject having or at risk ofcancer, and a second phase comprising an emulsifier, wherein thecomposition comprises no more than about 250 ppm of water by weight ofthe composition.
 19. The method of claim 18, wherein the compositioncomprises a liquid crystal structure comprising the phosphatidylcholine.20. The method of claim 18, wherein at least a portion of the liquidcrystal structure is multilamellar.
 21. The method of claim 18, whereinat least some of the phosphatidylcholine comprises apolyenylphosphatidylcholine.